Novel polyamine analog-amino acid conjugates useful as anticancer agents

ABSTRACT

Conjugates in which polyamine analogs are conjugated to an amino acid are provided, as well as compositions comprising these conjugates. Methods of using these conjugates as anticancer treatments are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of co-pending U.S.Provisional Patent Application Ser. No. 60/246,804, filed Nov. 8, 2000,and also of co-pending U.S. patent application No. 09/561,172 filed Apr.27, 2000, which in turn claims priority to U.S. Provisional PatentApplication Ser. Nos. 60/131,809, 60/131,779 and 60/131,842, all filedApr. 30, 1999. The entire contents of those applications are herebyincorporated by reference herein.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

[0002] Not applicable

TECHICAL FIELD

[0003] This invention relates to compositions in which a polyamine orpolyamine analog is conjugated to an amino acid. This invention alsorelates to uses of these conjugates as anticancer and antitumor agents.

BACKGROUND OF THE INVENTION

[0004] Cancer is one of the leading causes of death in the developedworld. Approximately one-quarter of the deaths in the United States in1997 were due to cancer, making it the second most common cause of deathafter heart disease. Accordingly, development of new and effectivetreatments for cancer is a high priority for health care researchers.

[0005] Cancer is often treated by using chemotherapy to selectively killor hinder the growth of cancer cells, while having a less deleteriouseffect on normal cells. Chemotherapeutic agents often kill rapidlydividing cells, such as cancer cells; cells which are dividing lessrapidly are affected to a lesser degree. Other agents, such asantibodies attached to toxic agents, have been evaluated for use againstcancers. These agents target the cancer cells by making use of acharacteristic specific to the cancer, for example, higher-than-normalrates of cell division, or unique antigens expressed on the cancer cellsurface.

[0006] Polyamines and polyamine analogs have been proposed asanti-cancer agents. Natural polyamines, e.g., spermidine, norspermidine,homospermidine, 1,4-diaminobutane (putrescine), and spermine, are simplealiphatic amines produced in eukaryotic cells by a highly regulatedmetabolic apparatus. Polyamine levels and the activity of the polyaminebiosynthetic apparatus tend to be high in dividing mammalian cells andlow in quiescent cells. Populations of cells depleted of their polyaminecontent stop growing and may die. Janne et al. (1978) A. Biochim.Biophys. Acta. 473:241 and Pegg et al. (1982) Ami. J. Cell. Phzysiol.243:212-221. Polyamines are reviewed in Morgan (1998) Methods. Mol.Biol. 79:3-30.

[0007] Several lines of evidence indicate that polyamines, particularlyspermidine, are required for cell proliferation: (i) they are found ingreater amounts in growing than in non-growing tissues; (ii) prokaryoticand eukaryotic mutants deficient in polyamine biosynthesis areauxotrophic for polyamines; and (iii) inhibitors specific for polyaminebiosynthesis also inhibit cell growth. Despite this evidence, theprecise biological role of polyamines in cell proliferation isuncertain. It has been suggested that polyamines, by virtue of theircharged nature under physiological conditions and their conformationalflexibility, might serve to stabilize macromolecules, such as nucleicacids, by anion neutralization. Hafner et al. (1979) J. Biol. Chem.254:12419; Pohjatipelto et al. (1981) Nature 293:475; Mamont et al.(1978) Biochem. Biophys. Res. Commun. 81:58; Bloomfield et al. (1981) inPolyamines in Biology and Medicine, Morris et al., Eds., Dekker, NewYork, pp. 183-205.

[0008] A treatment approach has been devised based on the observationthat increases in the polyamine pool suppress polyamine biosynthesis.Porter et al. (1988) in Advances in Enzyme Regulation, Pergamon Press,pp. 57-79. This approach attempts to identify polyamine analogs whichdown-regulate polyamine biosynthesis, but which do not perform thepolyamine functions required for cell growth. BESPM, a N-bis(ethyl)analog of spermine, has served as a model compound for this strategy.BESPM rapidly suppresses polyamine biosynthetic enzymes, depletesnatural polyamine pools, and inhibits cell growth in in vitro. Porter etal. (1987) Cancer Res. 47:2821-2825. In addition, BESPM suppressespolyamine uptake (Byers et al. (1990) J. Physiol. 142:460-467; andKramer et al. (1993) J. Cell. Physiol. 115:399-407), and thus minimizesthe ability of tumor cells to meet their polyamine requirement by takingthem up from their environment. BESPM and related analogs also inducethe polyamine metabolizing enzyme spermidine/spermineN¹-acetyltransferase (SSAT) in certain human carcinoma cell lines.

[0009] BESPM and other polyamine analogs have been used, or proposed foruse, in treating a large variety of diseases, including a number ofdifferent cancers. See International Patent Application WO 00/66587, andU.S. Pat. Nos. 5,889,061, 5,880,161, and 5,541,230. Polyamine analogsdemonstrated, for example, potent antitumor activity against severalmelanoma cell lines and tumors in vitro (Porter et al. (1991) CancerRes. 51:3715-3720; Shappell et al. (1992) Anticancer Res. 12:1083-1090)and in vivo using tumors growing as xenografts in athymic mice (Bernackiet al. (1992) Cancer Res. 52:2424-2430; Porter et al. (1993) Cancer Res.53:581-586). Potent antitumor activity of bis-ethyl spermine analogs hasalso been demonstrated for pancreatic cancer cell lines in vitro (Changet al. (1992) Cancer Chemother. Pharmacol. 30:183-188) and in vivo(Chang et al. (1992) Cancer Chemother. Pharmacol. 30:179-182). Polyamineanalogs have also been suggested for use in treating brain tumortherapy. Redgate et al. (1995) J. Neurooncol. 25:167-79. In addition tobeing useful against cancers of the brain, pancreas, and skin, polyamineanalogs are also useful against cancers of the bladder, bone, breast,colon, digestive tract, lung and ovaries. Chang et al. (1993) J. Urol.150:1293-7; Snyder et al. (1994) Anticancer Res. 14:347-56; Yuan et al.(1994) Biochem. Pharmacol. 47:1587-92; Davidson et al. (1993) CancerRes. 53:2071-5; Berchtold et al. (1998) J. Cell. Physiol. 174:380-6;Porter et al. (1988) Adv. Exp. Med Biol. 250:677-90; U.S. Pat. Nos.5,498,522 and 5,374,658. U.S. Pat. No. 5,498,522 presents the use ofspermindine/spermine N¹-acetyltransferase as a prognostic indicator ofthe efficacy of a polyamine analog against a malignant tumor.

[0010] Polyamine analogs have been used to treat cancer of the prostate)Mi et al. (1988) Prostate 34:51-60). Polyamines are produced in largeamounts by the prostate gland and are abundant in the seminal fluid(Herr et al. (1984) Cancer 53:12948). Polyamine analogs such as BE-4444,BE-373, and BE-333 are particularly effective in inhibiting prostatexenograft tumors in nude mice; see Zagaja et al. (1998) Cancer Chem.Pharm. 41:505-512; Jeffers et al. (1997) Cancer Chem. Pharm. 40:172-179;Feuerstein et al. (1991) J. Cell. Biochem. 46:37-47; and Marton et al.(1995) Ann. Rev. Pharm. Toxicol. 35:55-91.

[0011] Polyamines and their analogs can be administered alone or inconjunction with additional agents. For example, therapeutic polyaminescan be administered along with 1,3-bis (2-chloroethyl)-1-nitrosourea.U.S. Pat. No. 5,541,230. In treating cancer, polyamines can beco-administered with various cytotoxic agents, including antineoplasticvinca alkaloids, antibiotics, antimetabolites, and platinum coordinationcomplexes. U.S. Pat. No. 5,654,287.

[0012] It would be advantageous to develop novel polyamine analogs foruse as anti-cancer therapies. This invention describes

[0013] All references cited herein are hereby incorporated by referencein their entirety.

SUMMARY OF THE INVENTION

[0014] The invention provides compositions in which a polyamine orpolyamine analog is conjugated to one or more amino acids. The inventionalso provides methods for treatment of cancer by administering one ormore compositions of the invention. The polyamine or polyamine analogcan be combined with a pharmaceutically acceptable carrier or can bepresent as a pharmaceutically acceptable salt.

[0015] The polyamine or polyamine analog can be linked to an amino acidvia an amide linkage between a primary or secondary amine group of thepolyamine or polyamine analog, and the carboxy terminus of the aminoacid, for use in methods of the current invention. If the amino acidcontains more than one carboxyl group (for example, aspartatic acid orglutamic acid), the amide linkage can be to any of the carboxyl groupspresent in the amino acid. In one embodiment, the amino acid isconjugated to the polyamine or polyamine analog at one and only one ofthe exterior nitrogens of the polyamine or polyamine analog. In anotherembodiment, two amino acids are conjugated to the polyamine or polyamineanalog, one amino acid at each exterior nitrogen of the polyamine orpolyamine analog. The polyamine or polyamine analog can contain one ormore hydroxy groups, and can be linked to the carboxy terminus of one ormore amino acids by an ester linkage through the one or more hydroxygroups. In another embodiment, the polyamine or polyamine analog isconformationally restricted.

[0016] In another embodiment, the invention encompasses compositionscomprising a polyamine analog-amino acid conjugate of the formula:(M)-N(-E)-B-A-B-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH-E or(M)-N(-E)-B-A-B-NH-B-A-B-NH-B-A-B-NH-B-A-B-N(M)-E

[0017] wherein each M is independently an amino acid, each A isindependently selected from the group consisting of a single bond, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl,and C₃-C₆ cycloalkenyl; each B is independently selected from the groupconsisting of: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; and each Eis independently selected from the group consisting of H, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, andC₃-C₆ cycloalkenyl; and any salt or stereoisomer thereof.

[0018] In another embodiment, the invention encompasses compositionscomprising a polyamine analog-amino acid conjugate of the formula:(M)-N(-E)-B-A-B-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH-E or(M)-N(-E)-B-A-B-NH-B-A-B-NH-B-A-B-NH-B-A-B-N(M)-E

[0019] wherein each M is independently an amino acid, each A isindependently selected from the group consisting of a single bond, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆cycloalkenyl; each B is independently selected from the group consistingof: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; and each E isindependently selected from the group consisting of H, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, andC₃-C₆ cycloalkenyl; with the proviso that either at least one A moietyis selected from the group consisting of C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆ cycloalkenyl, or at leastone B moiety is selected from the group consisting of C₂-C₆ alkenyl; andany salt or stereoisomer thereof.

[0020] In another embodiment, the invention encompasses compositionscomprising a polyamine analog-amino acid conjugate of the formula:(M)-N(-E)-B-A-B-NH-B-A-B-NH-B-A-B-NH(-B-A-B-NH)_(x)-E or(M)-N(-E)-B-A-B-NH-B-A-B-NH- B-A-B-NH(-B-A-B-NH)_((x-1))-(-B-A-B-N(M))E

[0021] wherein each M is independently an amino acid, each A isindependently selected from the group consisting of: a single bond,C₆-C₂ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆cycloaryl, and C₃-C₆ cycloalkenyl; each B is independently selected fromthe group consisting of: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl;each E is independently selected from the group consisting of H, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl,and C₃-C₆ cycloalkenyl; and x is an integer from 2 to 16; and any saltor stereoisomer thereof.

[0022] In another embodiment, the invention encompasses compositionscomprising a polyamine analog-amino acid conjugate of the formula:(M)-N(-E)-B-A-B-NH-B-A-B-NH-B-A-B-NH(-B-A-B-NH)_(x)-E or(M)-HN-B-A-B-NH-B-A-B-NH- B-A-B-NH(-B-A-B-NH)_((x-1))-B-A-B-N(M)-E

[0023] wherein each M is independently an amino acid, each A isindependently selected from the group consisting of: a single bond,C₆-C₂ alkyl, C₂-C₆ alklenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆cycloaryl, and C₃-C₆ cycloalkenyl; each B is independently selected fromthe group consisting of: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl;each E is independently selected from the group consisting of H, C-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl,and C₃-C₆ cycloalkenyl; and x is an integer from 2 to 16; with theproviso that either at least one A moiety is selected from the groupconsisting of C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆cycloaryl, and C₃-C₆ cycloalkenyl, or at least one B moiety is selectedfrom the group consisting of C₂-C₆ alkenyl; and any salt or stereoisomerthereof.

[0024] In another embodiment, the invention encompasses compositionscomprising a polyarnine analog-amino acid conjugate of the formula:(M)E-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH(-B-A-B-NH)_(x)- E(M) or(M)E-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH(-B-A-B-NH)_(x)-E

[0025] wherein each M is independently an amino acid, wherein each A isindependently selected from the group consisting of: a single bond,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, andC₃-C₆ cycloalkenyl; each B is independently selected from the groupconsisting of: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; each E isindependently selected from the group consisting of C₁-C₆ alkyl, C₁-C₆alkanol, C₃-C₆ cycloalkanol, and C₃-C₆ hydroxyaryl, and the aminoacid(s) is linked to the polyamine analog via an ester linkage at the Egroup hydroxyl(s), with the proviso that each E bearing an amino acid isselected from C₁-C₆ alkanol, C₃-C₆ cycloalkanol, and C₃-C₆ hydroxyaryl;and x is an integer from 0 to 16; and any salt or stereoisomer thereof.

[0026] In another embodiment, the invention encompasses compositionscomprising a polyamine analog-amino acid conjugate of the formula:(M)E-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH(-B-A-B-NH)_(x)- E(M) or(M)E-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH(-B-A-B-NH)_(x)-E

[0027] wherein each M is independently an amino acid, wherein each A isindependently selected from the group consisting of: a single bond,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, andC₃-C₆ cycloalkenyl; each B is independently selected from the groupconsisting of: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; each E isindependently selected from the group consisting of C₁-C₆ alkyl, C₁-C₆alkanol, C₃-C₆ cycloalkanol, and C₃-C₆ hydroxyaryl, with the provisothat each E bearing an amino acid is selected from C₁-C₆ alkanol, C₃-C₆cycloalkanol, and C₃-C₆ hydroxyaryl; and the amino acid(s) is linked tothe polyamine analog via an ester linkage to at least one E grouphydroxyl(s); and x is an integer from 0 to 16; and any salt orstereoisomer thereof.

[0028] In another embodiment, the invention encompasses compositionscomprising a polyamine analog-amino acid conjugate of the formula:(M)-N(-E)-D-NH-B-A-B-NH-D-NH-E or (M)-N(-E)-D-NH-B-A-B-NH-D-N(M)-E

[0029] wherein each M is independently an amino acid; A is selected fromthe group consisting of C₂-C₆ alkynyl; each B is independently selectedfrom the group consisting of: a single bond, C₁-C₆ alkyl, and C₂-C₆alkenyl; each D is independently selected from the group consisting ofC₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆cycloalkenyl, and C₃-C₆ cycloaryl; and each E is independently selectedfrom the group consisting of H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆ cycloalkenyl; andany salt or stereoisomer thereof.

[0030] In another embodiment, the invention encompasses compositionscomprising a polyamine analog-amino acid conjugate of the formula:(M)-N(-E)-B-A-B-NH-F-NH-B-A-B-NH-E or(M)-N(-E)-B-A-B-NH-F-NH-B-A-B-N(M)-E

[0031] wherein each M is independently an amino acid; F is selected fromthe group consisting of C₁-C₆ alkyl; each A is independently selectedfrom the group consisting of: a single bond, C₁-C₆ alkyl; C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆cycloalkenyl;each B is independently selected from the group consistingof: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; and each E isindependently selected from the group consisting of H, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, andC₃-C₆ cycloalkenyl; and any salt or stereoisomer thereof.

[0032] In another embodiment, the invention encompasses compositionscomprising a polyamine analog-amino acid conjugate of the formula:(M)-N(-E)-B-A-B-NH-F-NH-B-A-B-NH-E or(M)-N(-E)-B-A-B-NH-F-NH-B-A-B-N(M)-E

[0033] wherein each M is indepedently an amino acid; wherein F isselected from the group consisting of C₁-C₆ alkyl; each A isindependently selected from the group consisting of: a single bond,C₁-C₆ alkyl; C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆cycloaryl, and C₃-C₆ cycloalkenyl; each B is independently selected fromthe group consisting of: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl;and each E is independently selected from the group consisting of H,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆cycloaryl, and C₃-C₆ cycloalkenyl; with the proviso that either at leastone A moiety is selected from the group consisting of C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆cycloalkenyl, or at least one B moiety is selected from the groupconsisting of C₂-C₆ alkenyl; and any salt or stereoisomer thereof.

[0034] In another embodiment, the invention encompasses compositionscomprising a polyamine analog-amino acid conjugate of the formula:

CH₃CH₂-N(R₁₀₀)-J-N(R₁₀₀)-CH₂CH₃,

[0035] where each R₁₀₀ is independently chosen from H, C₁-C₈ alkyl, andan amino acid, with the proviso that at least one R₁₀₀ be an amino acid;and where J is selected from {C₁-C₈ alkyl-[N(R₁₀₁)-(C₁-C₈ alkyl)]_(k)},where each R₁₀₁ is independently selected from H and C₁-C₈ alkyl, andwhere k is an integer between 0 and 15.

[0036] In further embodiments, both R₁₀₀ groups can be amino acids; oneand only one R₁₀₀ can be an amino acid, or one and only one R₁₀₀ is anamino acid and the other R₁₀₀ is H.

[0037] In another embodiment, the invention encompasses compositionscomprising a polyamine analog-amino acid conjugate of the formula:

CH₃CH₂-N(R₁₀₀)-(CH₂)₄-N(R₁₀₁)-(CH₂)₄-N(R₁₀₁)-(CH₂)₄-N(R₁₀₁)-(CH₂)₄N(R₁₀₀)-CH₂C₃

[0038] where each R₁₀₀ is independently chosen from H, C₁-C₈ alkyl, andan amino acid, with the proviso that at least one R₁₀₀ be an amino acid;and each R₁₀₁ is independently chosen from H and C₁-C₈ alkyl.

[0039] In another embodiment, the invention encompasses any one or anycombination of the foregoing embodiments, further comprising apharmaceutically acceptable excipient or carrier.

[0040] In another embodiment, the invention encompasses a method oftreating cancer in an individual, comprising administering to theindividual an effective amount of any one or any combination of theforegoing compositions. The individual can be a mammal, such as a human.

[0041] In any of the foregoing embodiments, when the compound containsone or more chiral centers, the invention encompasses any and all of thepure enantiomers and diastereomers of the compound, as well as racemicmixtures of the compounds, and/or any mixture or combination ofdiastereomers.

[0042] In any of the foregoing embodiments, the amino acid can chosenfrom the set of all amino acids; the set of all amino acids with eitheramide-containing or basic side chains; the set of glutamine, asparagine,lysine, ornithine, arginine, histidine, or citrulline; the set ofD-glutamine or L-glutamine; or from the single-member set ofL-glutamine; and all stereoisomers and salts thereof, unless astereoisomer is specifically excluded from the set.

[0043] In any of the foregoing embodiments, the amino acid can be chosenfrom the set of all amino acids, with the proviso that glutamine isexcluded; the set of all amino acids with either amide-containing orbasic side chains, with the proviso that glutamine is excluded; the setof asparagine, lysine, ornithine, arginine, histidine, or citrulline; orfrom the single-member set of D-glutamine; and all stereoisomers andsalts thereof, unless a stereoisomer is specifically excluded from theset.

[0044] In a subset of any of the foregoing embodiments, when the aminoacid is linked to a nitrogen group of the polyamine or polyamine analog,the amino acid can be an alpha-amino acid, linked to the polyamine orpolyamine analog via an amide linkage between the alpha-carboxyl groupof the amino acid and the nitrogen of the polyamine or polyamine analog.

BRIEF DESCRIPTION OF DRAWINGS

[0045]FIG. 1 illustrates synthetic methodology used to prepare polyamineor polyamine analog compounds useful in the invention.

[0046]FIG. 2 illustrates additional synthetic methodology used toprepare polyamine or polyamine analog compounds useful in the invention.

[0047]FIG. 3 illustrates additional synthetic methodology used toprepare polyamine or polyamine analog compounds useful in the invention.

[0048]FIG. 4 illustrates additional synthetic methodology used toprepare polyamine or polyamine analog compounds useful in the invention.

[0049]FIG. 5 illustrates additional synthetic methodology used toprepare polyamine or polyamine analog compounds useful in the invention.

[0050]FIG. 6 illustrates additional synthetic methodology used toprepare polyamine or polyamine analog compounds useful in the invention.

[0051]FIG. 7 illustrates additional synthetic methodology used toprepare polyamine or polyamine analog compounds useful in the invention.

[0052]FIG. 8A illustrates additional synthetic methodology used toprepare polyamine or polyamine analog compounds useful in the invention.

[0053]FIG. 8B illustrates additional synthetic methodology used toprepare polyamine or polyamine analog compounds useful in the invention.

[0054]FIG. 9 depicts Scheme 25, illustrating the synthesis of an aminoacid conjugated to a polyamine analog alchohol via an ester linkage.

[0055]FIG. 10 depicts Scheme 26, illustrating the synthesis of an aminoacid conjugated to various polyamine analogs via an amide linkage.

MODES FOR CARRYING OUT THE INVENTION

[0056] The present invention encompasses polyamine analog conjugates inwhich a polyamine analog is conjugated to an amino acid. The conjugatesof the invention are useful in treating cancer.

[0057] As discussed below, the polyamine analog can be any polyamineanalog, including, but not limited to, 1, 12-Me₂-SPM, SL-11027,SL-11028, SL-11029, SL-11033, SL-11034, SL-11037, SL-11038, SL-11043,SL-11044, SL-11047, SL-11048, SL-11050, SL-11090, SL-11091, SL11092,SL-11093, SL-11094, SL-11098, SL-11099, SL-11100, SL-11101, SL-11102,SL-11103, SL-11104, SL-11105, SL-11108, SL-11114, SL-11118, SL-11119,SL-11121, SL-11122, SL-11123, SL-11124, SL-11126, SL-11127, SL-11128,SL-11129, SL-11130, SL-11132, SL-11133, SL-11134, SL-11136, SL-11137,SL-11141, SL-11144, SL-11150, SL-11201, and SL-11202. Preferably, thepolyamine analog is conformationally restricted.

[0058] Definitions

[0059] By “polyamine analog” is meant an organic cation structurallysimilar but non-identical to polyamines such as spermine and/orspermidine and their precursor, diamine putrescine. By a “polyamine” ismeant any of a group of aliphatic, straight-chain amines derivedbiosynthetically from amino acids; polyamines are reviewed in Marton etal. (1995) Ann. Rev. Pharm. Toxicol. 35:55-91. Polyamines cadaverine andputrescine are diamines produced by decarboxylation of lysine orornithine, respectively. Putrescine is converted to spermidine, andspermidine to spermine, by the addition of an aminopropyl group. Thisgroup is provided by decarboxylated S-adenosyl methionine. Polyamineanalogs, which can be branched or un-branched, include, but are notlimited to, BE-4444 [1,19-bis(ethylamino)-5,10,15-triazanonadecane];BE-333 [N1,N11-diethylnorspermine; DENSPM;1,11-bis(ethylarnino)-4,8-diazaundecane; thermine; Warner-Parke-Davis];BE-33 [N1,N7-bis (ethyl) norspermidine]; BE-34 [N1,N8-bis (ethyl)spermidine]; BE44 [N1,N9-bis (ethyl) homospermidine]; BE-343 [N1,N12-bis(ethyl) spermine; diethylspermine-N1-N12; DESPM]; BE-373[N,N′-bis(3-ethylamino) propyl)-1,7-heptane diamine, Merrell-Dow]; BE444[N1,N14-bis (ethyl) homospermine; diethylhomospermine-N1-N14); BE-3443[1,17-bis (ethylamino)4,9,14triazaheptadecane]; BE4334 [1,17-bis(ethylamino)-5,9,13-triazaheptadecane]; 1,12-Me₂-SPM[1,12-dimethylspermine]; various polyarrine analogs disclosed in WO98/17624 and U.S. Pat. No. 5,889,061; and the various novel polyamineanalogs illustrated in the Figures and described herein, including, butnot limited to, compounds designated SL-11027, SL-1 1028, SL-11029,SL11033, SL-11034, SL-11037, SL111038, SL-11043, SL-11044, SL-11047,SL-11048, SL-11050, SL-11090, SL-11091, SL-11092, SL-11093, SL-11094,SL-11098, SL-11099, SL-11100, SL-11101, SL-11102, SL-11103, SL-11104,SL-11105, SL-11108, SL-11114, SL-11118, SL-11119, SL-11121, SL-11122,SL-11123, SL-11124, SL-11126, SL-11127, SL-11128, SL-11129, SL-11130,SL-11132, SL-11133, SL-11134, SL-11136, SL-11137, SL-11141, SL-11144,SL-11150, SL-11201, and SL-11202. Additional polyamine analogs usefulfor this invention are known in the art, such as O'Sullivan et al.(1997) Bioorg. Med Chem. 5:2145-2155; and Mukhopadhyaya et al. (1995)Exp. Parasit. 81:39-46; and U.S. Pat. No. 4,935,449.

[0060] By “conformationally restricted” is meant that, in a polyamineanalog, at least two amino groups are locked or limited in spatialconfiguration relative to each other. The relative movement of two aminogroups can be restricted, for example, by incorporation of a cyclic orunsaturated moiety between them (exemplified, but not limited to, aring, such as a three-carbon ring, four-carbon ring, five-carbon-ring,six-carbon ring, or a double or triple bond, such as a double or triplecarbon bond). Groups restricting conformational flexibility by means ofsteric hindrance, yet structurally favorable to the anti-proliferativeeffects, can also be used according to the invention. A“conformationally restricted” polyamine analog can comprise at least twoamino groups which are conformationally restricted relative to eachother, but can also further comprise amino groups which are notconformationaUy restricted relative to each other. Flexible moleculessuch as spermine and BE444 can have a myriad of conformations and aretherefore not conformationally restricted.

[0061] For the purposes of this invention, amino acids are defined toinclude the twenty genetically-encoded amino acids (the twentygenetically encoded amino acids include the imino acid proline), othernaturally-occurring amino acids, such as ornithine and citrulline,non-naturally occurring amino acids, and all stereoisomers and saltsthereof. One embodiment of the invention utilizes the subset of theamino acids which have either amide-containing or basic side chains(amino acids containing basic side chains include, but are not limitedto, amino acids with amino-containing side chains such as homolysine,amino acids with ureido-containing side chains such as citrulline, aminoacids containing imidazole side chains such as histidine, or amino acidscontaining guanidino-containing side chains, such asalpha-amino-beta-guanidino propionic acid), and all stereoisomers andsalts thereof. Another embodiment of the invention utilizes thefollowing subset of amino acids: glutamine, asparagine, lysine,ornithine, arginine, histidine, or citrulline, and all stereoisomers andsalts thereof. Another embodiment of the invention utilizes thefollowing subset of amino acids: D-glutamine or L-glutamine, and allsalts thereof. In another embodiment, the amino acid is L-glutamine, andall salts thereof. In another embodiment of the invention, the aminoacid is beta-alanine (3-amino propionic acid, H₂NCH₂CH₂COOH).

[0062] The terms “peptide,” “polypeptide,” “oligopeptide,” and the likeare used interchangeably herein to refer to any polymer of amino acidresidues of any length. The peptide polymer can be linear or non-linear(e.g., branched), it can comprise modified amino acids or amino acidanalogs, and it can be interrupted by chemical moieties other than aminoacids. The terms also encompass an amino acid polymer that has beenmodified naturally or by intervention; for example, by disulfide bondformation, glycosylation, lipidation, acetylation, phosphorylation, orany other manipulation or modification, such as conjugation with alabeling or bioactive component.

[0063] By “conjugation” is meant the process of forming a covalentlinkage, with or without an intervening linker, between two moieties,such as a polyamine analog and a amino acid moiety. The conjugation canbe performed by any method known in the art, such as those described inWong, Chemistry of Protein Conjugation and Cross-linking, 1991, CRCPress, Boca Raton, and described herein. Suitable methods include usingstrategies incorporating protecting groups such as thet-butyloxycarbonyl (BOC) protecting group (reagents for introducing theBOC group are available from Sigma, St. Louis, Mo., and othersuppliers). Other suitable protecting groups which can be used in theconjugation reactions are described in Greene et al., Protective Groupsin Organic Synthesis, 2nd Edition, 1991, Wiley, N.Y. By “conjugate” ismeant a chemical entity comprising two moieties which are covalentlylinked.

[0064] An “amino-capping group” or “amino-terminal capping group” is agroup that covalently links to an amino group. Examples of amino-cappinggroups include, but are not limited to, 4-morpholinocarbonyl, acetyl,and trifluoroacetyl. An “amino-protecting group” or “amino-terminalprotecting group” is a group that can be selectively removed from anamino group of a molecule without affecting the remainder of themolecule. Examples of amino-protecting groups include, but are notlimited to, t-butyloxycarbonyl (BOC), 9-fluorenylmethoxycarbonyl (FMOC),benzyloxycarbonyl (CBZ ), t-butyldimethylsilyl (TBDIMS), or suitablephotolabile protecting groups such as 6-nitroveratryloxy carbonyl (Nvoc)and the like.

[0065] An “exterior nitrogen” or “exterior amino group” of a polyamineor polyamine analog is a nitrogen (amino) group which is flanked by onlyone other nitrogen group, while an “interior nitrogen” or “interioramino group” of a polyamine or polyamine analog is a nitrogen (amino)group which is flanked by two other nitrogen (amino) groups. Forexample, in a polyamine analog of the formula

R₁-N¹H-R₂-N²H-R₃-N³H- . . . -R_((n-1))-N^(n-1))H-N^(n)H-R_(n),

[0066] where n is an integer, the nitrogens designated as N¹ and N^(n)are the “exterior nitrogens” or “exterior amino groups,” inasmuch asthey are flanked by only one other nitrogen group, while N², N³, etc.,through N^((n-1)) are “interior nitrogens” or “interior amino groups,”flanked by two other nitrogen (amino) groups.

[0067] An “individual” is a vertebrate, preferably a mammal, morepreferably a human.

[0068] An “effective amount,” “therapeutic amount,” or “therapeuticallyeffective amount” is an amount sufficient to effect beneficial ordesired clinical results. An effective amount can be administered in oneor more administrations. For purposes of this invention, an effectiveamount of a polyamine analog conjugate is an amount that is sufficientto cure, palliate, ameliorate, stabilize, reverse, slow or delay theprogression of the disease state, or the symptoms of the disease state.

[0069] As used herein, “treatment” is an approach for obtainingbeneficial or desired clinical results, including, but not limited to,the suppression of viral growth. For purposes of this invention,beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease,stabilization (i.e., not worsening) of state of disease, prevention ofspread (e.g., contagion) of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state,improvement in quality or enjoyment of life, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment.

[0070] “Palliating” a disease means that the extent and/or undesirableclinical manifestations of a disease state are lessened and/or timecourse of the progression is slowed or lengthened, as compared to notadministering polyamine analog conjugates of the present invention.

[0071] Polyamine Analogs Useful In The Invention

[0072] One embodiment of the present invention encompasses a polyamineanalog conjugated to an amino acid. Other aspects of the inventionencompass compositions comprising these conjugate(s). Non-limitingexamples of polyamine analogs which can be used are described below.

[0073] Conformationally Restricted Polyamine Analogs

[0074] Polyamine analogs which can be used in the invention include anypolyamine analog that has a pendant amino, hydroxyl, or thiol groupwhich can be conjugated to the C-terminus of the amino acid moiety in anamide linkage, ester linkage or thioester linkage, respectively, andexamples are provided in the summary of the invention, the definition of“polyamine analogs” and in the synthetic schemes. Polyamine analogs usedin the present invention can be conformationally restricted.

[0075] Schemes 1-25 (see FIGS. 1-10) depict syntheses of variouspolyamine analogs which can be used in the invention. Examples ofpolyamine analogs which can be used in the invention are also given inU.S. Pat. Nos. 5,889,061 and 5,627,215, which describe tetraaminopolyamine analogs. The synthesis of the polyamine analogs of thosepatents can be modified to introduce an amino-protecting group on theexterior nitrogens (i.e., representing the tetraamine asR₁-N¹H-R₂-N²H-R₃-N³H-R₄-N⁴H-R₅, the nitrogens designated as N¹ and N⁴are the “exterior” nitrogens, inasmuch as they are flanked by only oneother nitrogen group, while N² and N³ are “interior” nitrogens, flankedby two other nitrogen groups) in place of the group that wouldordinarily be attached at that point (in this example, a protectinggroup would be used instead of R₁ or R₅), and can be cleaved to yield aprimary amino group at one of the exterior nitrogens, while maintainingamino-protecting groups on the other exterior nitrogen and the interiornitrogens. Scheme 26 of FIG. 10 depicts such a strategy of establishinga protecting group regimen which allows one of the exterior amino groupsto be selectively deprotected, while maintaining the amnino-protectinggroups on the other exterior amino group and the interior amino groups.Examples of differential protection regimens of polyamine or polyamineanalogs are also given in Fiedler et al. (1993) Helv. Chim. Acta76:1511-1519 and Iwata et al. (1989) Bull. Chem. Soc. Japan62:1102-1106. TABLE 1 No. Structure SL-11027

SL-11028

SL-11029

SL-11033

SL-11034

SL-11035

SL-11036

SL-11037

SL-11038

SL-11043

SL-11044

SL-11047

SL-11048

SL-11050 BnNH(CH₂)₄NHBn.2HCl SL-11061EtNH(CH₂)₄NH(CH₂)₄NH(CH₂)₄NH(CH₂)₄-NHEt.5HCl SL-11090

SL-11091

SL-11092

SL-11093

SL-11094

SL-11098

SL-11099

SL-11100

SL-11101

SL-11102

SL-11103

SL-11104

SL-11105

SL-11108

SL-11114

SL-11118

SL-11119

SL-11121

SL-11122

SL-11123

SL-11124

SL-11126

SL-11127

SL-11128

SL-11129

SL-11130

SL-11132

SL-11133

SL-11134

SL-11135

SL-11136

SL-11137

SL-11141

SL-11143

SL-11144

SL-11150

SL-11155

SL-11157

SL-11158

SL-11159

SL-11160

[0076] Conjugation Of Polyamine Analogs To The Amino Acid Moiety

[0077] Any method known in the art can be used to conjugate (i.e., link)the amino acid to the polyamine analog, including, but not limited to,those disclosed herein. Suitable methods include using strategiesincorporating protecting groups such as the t-butyloxycarbonyl (BOC)protecting group (reagents for introducing the BOC group are availablefrom Sigma, St. Louis, Mo., and other suppliers). Other suitableprotecting groups which can be used in the conjugation reactions aredescribed in Greene et al., Protective Groups in Organic Synthesis 2ndEdition, 1991, Wiley, N.Y. Coupling chemistry is described in Bodanszky,M. and Bodanszky, A., The Practice of Peptide Synthesis, New York:Springer-Verlag, 1984; Bodanszky, M., Principles of Peptide Synthesis,2nd Edition, New York: Springer-Verlag, 1993; Bodanszky, M., PeptideChemistry: A Practical Textbook, 2nd Edition, New York: Springer Verlag,1993; Fmoc Solid Phease Peptide Synthesis : A Practical Approach(Practical Approach Series) (Weng Chan and Peter D. White, Eds.) (OxfordUniversity Press, 2000), and Solid Phase Peptide Synthesis (John M.Stewart and Janis D. Young; Pierce Chemical Co., Rockford Ill., 1984).When alpha-amino acids are used, the amino acids are preferentiallycoupled via the alpha-COOH group of the C-terminal amino acid, althoughother linkages are possible, depending on the amino acid (e.g., thegamma-carboxyl group of a glutamic acid residue can be used forlinkage). When amino acids other than alpha-amino acids are used, ifonly one carboxyl group is present, the linkage is preferably to thatcarboxylic acid group of the amino acid; when more than one carboxylicacid group is present in a non-alpha-amino acid, any availablecarboxylic acid group can be used for the coupling. When a polyamine orpolyamine analog is coupled, the linkage will be via an amino group ofthe polyamine (i.e., an amide linkage); when a polyamine analog alcoholis conjugated, the linkage can be via either an amino group or a hydroxygroup of the polyamine analog alcohol (i.e., an amide linkage or esterlinkage, respectively). The amino acid is preferably coupled to anexterior nitrogen. When an ester linkage to a polyamine analog is used,the amino acid is preferably coupled to a terminal hydroxy group.

[0078] Therapeutic Use Of Polyamine- and Polyamine Analog-amino AcidConjugates

[0079] Polyamine- and polyamine analog-amnino acid conjugates of thepresent invention are useful for treatment of a variety of diseasescaused by uncontrolled proliferation of cells, including cancer,particularly prostate cancer and other cancers. The conjugates are usedto treat mammals, preferably humans.

[0080] In order to evaluate the efficacy of a particular polyamine- orpolyamine analog-amino acid conjugate for a particular medicinalapplication, the compounds can be first tested against appropriatelychosen test cells in vitro. In a non-limiting example, the conjugatescan be tested against tumor cells, for example, prostate tumor cells.Exemplary experiments can utilize cell lines capable of growing inculture as well as in vivo in athymic nude mice, such as LNCAP.Horoszewicz et al. (1983) Cancer Res. 43:1809-1818. Culturing andtreatment of carcinoma cell lines, cell cycle and cell deathdeterminations based on flow cytometry; enzyme assays including ODC,SAMDC and SSAT activities; and high pressure liquid chromatographydetection and quantitation of natural polyamines and polyamine analogsare described in the art, for example, Mi et al. (1998) Prostate34:5160; Kramer et al. (1997) Cancer Res. 57:5521-27; and Kramer et al.(1995) J. Biol. Chem. 270:2124-2132. Evaluations can also be made of theeffects of the conjugates on cell growth and metabolism.

[0081] Analysis begins with IC₅₀ determinations based on dose-responsecurves ranging from 0.1 to 1000 μM performed at 72 hr. From thesestudies, conditions can be defined which produce about 50% growthinhibition and used to: (a) follow time-dependence of growth inhibitionfor up to 6 days, with particular attention to decreases in cell number,which may indicate drug-induced cell death; (b) characterize conjugateeffects on cell cycle progression and cell death using flow cytometry(analysis to be performed on attached and detached cells); (c) examineconjugate effects on cellular metabolic parameters. Polyamnine- andpolyamine analog-amino acid conjugate effects can be normalized tointracellular concentrations (by HPLC analysis), which also provide anindication of their relative ability to penetrate cells. Markeddifferences in conjugate uptake can be further characterized by studyingconjugate ability to utilize and regulate the polyamine transporter, asassessed by competition studies using radiolabeled spermidine, aspreviously described in Mi et al. (1998). Polyamine- and polyamineanalog-amino acid conjugates could also enter the cells by a diffusionmechanism.

[0082] In vivo testing of polyamine- and polyamine analog-amino acidconjugates

[0083] Polyamine- and polyamine analog-amino acid conjugates found tohave potent anti-proliferative activity in vitro towards culturedcarcinoma cells can be evaluated in in vivo model systems. The firstgoal is to determine the relative toxicity of the conjugates innon-tumor-bearing animals, such as DBA/2 mice. Groups of three animalseach can be injected intraperitoneally with increasing concentrations ofa conjugate, beginning at, for example, 10 mg/kg. Toxicity as indicatedby morbidity is closely monitored over the first 24 hr. Awell-characterized polyamine analog, such as BE-333, can be used as aninternal standard in these studies, since a data base has already beenestablished regarding acute toxicity via a single dose treatmentrelative to chronic toxicity via a daily ×5 d schedule. Thus, in thecase of new compounds, single dose toxicity relative to BE-333 is usedto project the range of doses to be used on a daily ×5 d schedule.

[0084] After the highest tolerated dosage on a daily ×5 d schedule isdeduced, antitumor activity is determined. Typically, tumors can besubcutaneously implanted into nude athymic mice by trocar and allowed toreach 100-200 mm³ before initiating treatment by intraperitonealinjection daily ×5 d. Most conjugates can be given in a range between 10and 200 mg/kg. Conjugates can be evaluated at three treatment dosageswith 10-15 animals per group (a minimum of three from each can be usedfor pharmacodynamic studies, described below). Mice can be monitored andweighed twice weekly to determine tumor size and toxicity. Tumor size isdetermined by multi-directional measurement from which volume in mm³ iscalculated. Tumors can be followed until median tumor volume of eachgroup reaches 1500 mm³ (i.e., 20% of body weight), at which time theanimals can be sacrificed. Although the initial anti-tumor studiesfocuses on a daily ×5 d schedule, constant infusion can be performed viaAlzet pump delivery for 5 days since this schedule dramatically improvesthe anti-tumor activity of BE-333 against A549 human large cell hungcarcinoma. Sharma et al. (1997) Clin. Cancer Res. 3:1239-1244. Inaddition to assessing anti-tumor activity, free conjugate levels intumor and normal tissues can be determined in test animals.

[0085] Methods of adimistration of polyamine- and polyamine analog-aminoacid conjugates

[0086] The polyamine- and polyamine analog-amino acid conjugates of thepresent invention can be administered to a mammalian, preferably human,subject via any route known in the art, including, but not limited to,those disclosed herein. Preferably administration of the conjugates isintravenous. Other methods of administration include but are not limitedto, oral, intrarterial, intratumoral, intramuscular, topical,inhalation, subcutaneous, intraperitoneal, gastrointestinal, anddirectly to a specific or affected organ. The novel polyamine analogsdescribed herein are administratable in the form of tablets, pills,powder mixtures, capsules, granules, injectables, creams, solutions,suppositories, emulsions, dispersions, food premixes, and in othersuitable forms. The compounds can also be administered in liposomeformulations. The compounds can also be administered as prodrugs, wherethe prodrug undergoes transformation in the treated subject to a formwhich is therapeutically effective. Additional methods of administrationare known in the art.

[0087] The pharmaceutical dosage form which contains the compoundsdescribed herein is conveniently admixed with a non-toxic pharmaceuticalorganic carrier or a non-toxic pharmaceutical inorganic carrier. Typicalpharmaceutically-acceptable carriers include, for example, mannitol,urea, dextrans, lactose, potato and maize starches, magnesium stearate,talc, vegetable oils, polyalkylene glycols, ethyl cellulose,poly(vinylpyrrolidone), calcium carbonate, ethyl oleate, isopropylmyristate, benzyl benzoate, sodium carbonate, gelatin, potassiumcarbonate, silicic acid, and other conventionally employed acceptablecarriers. The pharmaceutical dosage form can also contain non-toxicauxiliary substances such as emulsifying, preserving, or wetting agents,and the like. A suitable carrier is one which does not cause anintolerable side effect, but which allows the conjugates to retain itspharmacological activity in the body. Formulations for parenteral andnonparenteral drug delivery are known in the art and are set forth inRemington's Pharmaceutical Sciences, 18th Edition, Mack Publishing(1990). Solid forms, such as tablets, capsules and powders, can befabricated using conventional tableting and capsule-filling machinery,which is well known in the art. Solid dosage forms, including tabletsand capsules for oral administration in unit dose presentation form, cancontain any number of additional non-active ingredients known to theart, including such conventional additives as excipients; dessicants;colorants; binding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinylpyrollidone; fillers, for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricants, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants, for example potato starch; or acceptable wettingagents such as sodium lauryl sulphate. The tablets can be coatedaccording to methods well known in standard pharmaceutical practice.Liquid forms for ingestion can be formulated using known liquidcarriers, including aqueous and non-aqueous carriers, suspensions,oil-in-water and/or water-in-oil emulsions, and the like. Liquidformulations can also contain any number of additional non-activeingredients, including colorants, fragrance, flavorings, viscositymodifiers, preservatives, stabilizers, and the like. For parenteraladministration, conjugates can be administered as injectable dosages ofa solution or suspension of the compound in a physiologically acceptablediluent or sterile liquid carrier such as water or oil, with or withoutadditional surfactants or adjuvants. An illustrative list of carrieroils would include animal and vegetable oils (peanut oil, soy bean oil),petroleum-derived oils (mineral oil), and synthetic oils. In general,for injectable unit doses, water, saline, aqueous dextrose and relatedsugar solutions, and ethanol and glycol solutions such as propyleneglycol or polyethylene glycol are preferred liquid carriers. Thepharmaceutical unit dosage chosen is preferably fabricated andadministered to provide a final concentration of drug at the point ofcontact with the cancer cell of from 1 μM to 10 mM, from 1 μM to 1 mM,from 1 μM to 100 μM, from 1 μM to 10 μM, from 1 nM to 1 μM, from 1 nM to100 nM, or from 1 nM to 10 nM. The optimal effective concentration ofpolyamine- and polyamine-amino acid conjugates can be determinedempirically and will depend on the type and severity of the disease,route of administration, disease progression and health and mass or bodyarea of the patient. Such determinations are within the skill of one inthe art. Polyamine- and polyamine-amino acid conjugates can beadministered as the sole active ingredient, or can be administered incombination with another active ingredient, including, but not limitedto, cytotoxic agents, antibiotics, antimetabolites, nitrosourea, vincaalkaloids, polypeptides, antibodies, cytokines, etc.

[0088] The following examples are provided to illustrate, but not limit,the invention.

EXAMPLES Synthesis of conformationally-restricted polyamine analogs a)spermine and homospermine analogs containing a conformationalrestriction

[0089] Scheme 2 exemplifies a N^(α), N^(ω)-bisethyl homospermine analog7 containing a central trayis-unsaturated bond. Amide 4 was prepared asdescribed in Scheme 1 by alkylation of amide 1 with bromobutyronitrileto give 2, followed by reduction of the nitrile to the amine 3 that wasmesitylsulfonated to 4. Trans-allylic diester 5 was used to alkylateamide 4 and the tetramide 6 was obtained. Deprotection gave thetrans-tetramide 7 (Scheme 2). Introduction of a triple bond in thebutane segment of homospermine also reduces its mobility. This wasachieved by starting with the butyne diester 8 and following thesequence of reactions outlined above (Scheme 3). Schemes 15-20 arefurther examples of the synthesis of polyamine spermine and homospermineanalogs of this type.

b) Synthesis of pentamines with conformational restrictions.

[0090] Schemes 4-14 are outlines of the syntheses of conformationallyrestricted pentamines. Scheme 4 depicts the reaction ofcis-1-chloro-4-phthalimido butene with amide 1 to give 11.Hydrazinolysis of 11 gave 12 which was amidated to 13. Reaction of thelatter with 1,4-diiodobutane gave 14, while reaction with equimolaramounts of cis-1,4-dichlorobutene gave 15.

[0091] Amide 4 was alkylated with either 4-chlorobutyronitrile to give16 or with cis-1,4-dichlorobutene to give 19. Nitrile 16 was reducedwith hydrogen over Ni Raney to the amine 17 and the latter transformedin to the amide 18 (Scheme 5). Condensation of 18 with the chloroalkylintermediate 15 gave the pentamaide 20 that was deprotected to thepentamine 21 (Scheme 6). Condensation of 18 with the iodoalkylderivative 14 gave 22 that was deprotected to the pentamine 23 (Scheme7). Condensation of 18 and 19 gave pentamide 24 that was deprotected tothe pentamine 25 (Scheme 8). Using 14 as the alkylating agent,mesitylenesulfonamide was dialkylated to give 26, and the latterdeprotected to give 27 (Scheme 9). The analogous reaction carried outusing 15 as alkylating agent, gave 28 and after deprotection led to thepentamine 29 (Scheme 10).

[0092] Alkylation of mesitylenesulfonamide with 19 gave the pentamide30, which was deprotected to 31 (Scheme 11). When 19 was used toalkylate an equimolar amount of mesitylenesulfonamide then 32 wasobtained. Alkylation of 32 with 14 gave 33, that was deprotected to give34 (Scheme 12). When the chloroalkyl intermediate 15 was used toalkylate one equivalent of mesitylenesulfonamide, then the triamide 35was obtained. Reaction of 35 with 14 gave 36 which was then deprotectedto 37 (Scheme 13). Condensation of 35 and 19 gave the pentamide 38 thatwas deprotected to 39 (Scheme 14). The above mentioned Schemes describethe synthesis of cis-compounds. The same synthetic methodology can beused to obtain the trans-isomers, or cis and traits bonds in differentsegments within the same molecule.

c) Polyamine analog with diamidine substituents.

[0093] A new class of polyamine analogs is shown in Scheme 21. Theyderive from 1,4-dibenzylputrescine, 1,5-dibenzylcadaverine, and1,6-dibenzylhexanediamine. They are diamidine derivatives, where thediamidine residues are carrier groups that have been shown to beefficient in the transport of drugs into different protozoa. The generalprocedure of synthessis was based on the condensation of4-cyanobenzaldehyde with the diaminoalkanes to give the Schiff bases,followed by reduction in situ to the corresponding dinitriles 68. Thelatter were converted to the diamidines 69 through their iminoethers.

d) Synthesis of oligoaniines.

[0094] Scheme 22 describes the synthesis of a N-2 hydroxyethylderivative of a pentamine such as 75. Starting with 18, alkylation with4-bromobutyronitrile gave 70. Reduction of the nitrile of 70 andmesitylenesulfonylation of the resulting amino group gave 71. It wasalkylated again with 4-bromobutyronitrile to give 72, and again reducedand mesitylsulfonylated to give 73. The latter was then alkylated withthe benzyl ester of 2-bromoethanol to give 74. Treatment withhydrobromic acid in acetic acid cleaved both the mesitylene sulfonylprotecting groups and the benzyl ether residue to give 75.

[0095] Scheme 23 reports the synthesis of a trans-decamine 77 and of acis-decamine 79. Starting with the pentamiide 73 (Scheme 22) and byreaction with trans-diester 5 (Scheme 2) the decamide 76 was prepared,which on deprotection gave 77 as a decahydrochloride. In an analogousmanner, by condensation of 73 with the cis-1,4-dimesityleneoxy-2-butene,the decamide 78 was prepared, which on deprotection gave 79 as adecahydrochloride.

[0096] Scheme 24 outlines the synthesis of a N-2 hydroxyethyltrans-decamine 92 and a cis-2-hydroxyethyl decamine 95. The procedurerepeats almost all the procedures described in the foregoing schemes.The synthesis of 80 proceeded by alkylating BOC-mesitylenesulfonamidewith the benzyl ester of 2-bromoethanol. Cleavage of the BOC protectinggroup leads to 81, alkylation with 4-bromobutyronitrile then gave 82,and after reduction of the nitrile group and reaction with mesitylenesulfonyl chloride the diamide 83 was obtained. Again, alkylation with4-bromobutyronitrile led to 84, reduction and mesitylsulfonylation gave85, alkylation of 85 gave 86, reduction and mesitylsulfonylation gave87, and alkylation, reduction and mesitylsulfonylation performed on 87gave 89. Alkylation of 73 with trans-1,4-dibromo-2-butene gave 90.Alkylation of 89 with 90 gave 91, which after deprotection gave thetrans-ω-hydroxy-decamine 92. Alkylation of 73 withcis-1,4-dichloro-2-butene gave 93. Alkylation of 89 with 93 gave 94.Deprotection of 94 gave the cis-ω-hydroxy-decamine 95, isomeric with 92.

e) Sythesis of oligoamine-aminio acid conjugate 97.

[0097] Scheme 25 outlines the synthesis of an amino acid derivative of75 (SL-11141). Starting with 74, hydrogenolysis leads to 96, that isthen is then esterified with N-BOC-glutamine to 97.

(f) Synthesis of polyamtine analog conjugates of azino acids

[0098] Scheme 26 outlines the synthesis of polyamine analog conjugatesof the polyamine analogs corresponding to SL-11047, SL-11101, orBE4-4-4-4 to give the conjugates 112, 113, and 114. The polyamine analogintermediates are constructed as follows. Chloride 100 is condensed with46 to give 101. The phthalimido group is cleaved by hydrazynolysis togive 102, and the latter is mesitylated to 103 This amide is againalkylated with 104 to give 105. The mesitylene sulfonyl groups of 105are then cleaved and 106 is obtained. It is protected using (BOC)₂O, andthe resulting 102 is deprotected by hydrazynolysis to give the polyamineanalog moiety of 112. In tandem, the known 74 (Scheme 17) was alkylatedwith 108 to give 109. Cleavage of the mesitylenesulfonyl groups gave110. The free amino groups were reprotected with (BOC)₂O to give 111.Cleavage of the phthalimido residue via hydrazinolysis using a procedureanalogous to that for compound 12 below gave the aminopolyamine analogintermediate for the synthesis of 114.

[0099] In one embodiment, both exterior nitrogens of the polyamineanalog-amino acid conjugates have an ethyl group as one of theirsubstituents. In the synthesis outlined above, one exterior nitrogenalready bears an ethyl group; the synthesis can be readily modified toadd an ethyl group to the other exterior nitrogen. For example, thephthalimido group on the intermediate 111 in Scheme 26 is removed usinghydrazinolysis using a procedure analogous to that for compound 12 belowto yield CH₃CH₂N(Boc)[(CH₂)₄N(Boc)]₃(CH₂)₄NH₂. This primary aminecontaining compound is then reacted with an ethyl halide (e.g., ethylbromide, ethyl iodide, ethyl chloride) or another reactive ethylationagent, e.g., ethyl mesylate. The alkylating reagent is generally addeddropwise or in small portions to an excess of the primary amine to avoidfurther alkylation of the desired product,CH₃CH₂N(Boc)[(CH₂)₄N(Boc)]₃(CH₂)₄NHCH₂CH₃. This product is then reactedwith an amino acid, e.g., Nα-Boc-glutamine, Nα-Boc-asparagine,N-α,ε-Boc-lysine, or other desired amino acid, suitably protected toreact at its α-carboxylic acid functionality (or, if desired, at theside chain acid functionality of glutamic acid and aspartic acid).Dicyclohexylcarbodiimide, diisopropylcarbodiimide, HBTU, HATU, or othercoupling reagents and protocols well-known in the art can be used tocouple the amino acid to the polyamine analog. The progress of thereaction can be monitored by HPLC or other methods.

[0100] Coupling of amino acids to any or all other amino groups of apolyarnine or polyamine analog can be carried out by using appropriatelyprotected subunits during synthesis. The general scheme involves usingprotecting groups which can be removed independently of each other;methods of differentially protecting compounds are shown in, e.g.,Scheme 26.

EXAMPLES Example 1. Synthesis Of Polyamine Analog Compounds

[0101] Compound 2: NaH (80%, 1.08 g, 36 mmol) was added to a solution ofamide 1 (6.81 g, 30 mmol) in DMF (50 ml) in an ice-water bath under N₂.The mixture was stirred for 1 h and a solution of 4-bromobutyronitrile(4.88 g, 33 mmol) in DMF (10 ml) was added in portions. The mixture wasstirred over night at 75° C. The solvent was distilled off, the residuetaken up in chloroform washed with a saturated solution of ammoniumchloride, dried (Na₂SO₄) and evaporated. The residue was purifid byflash chromatography on silica gel (hexane/ethyl acetate 3:1) to yield8.0 g (90%) of 2 as a colorless oil. ¹H-NMR (CDCl₃) δ1.05 (t, 3H), 1.90(m, 2H), 2.30 (b, m, 5H), 2.60 (s, 6H), 3.20 (q, 2H), 3.35 (t, 2H), 6.95(s, 2H); ¹³C-NMR (CDCl₃): δ12.50, 20.61, 22.43, 23.60, 31.05, 36.12,40.39, 43.78, 118.62, 131.79, 132.67, 139.71, 142.41. MS-EI (m/z) 294(M⁺).

[0102] Compound 4: Nitrile 2 (7.8 g, 27 mmol) was dissolved in a mixtureof ethanol (150 ml) and concentrated hydrochloric acid (1.5 ml). PtO₂was added (700 mg) and the mixture was hydrogenated at 50 psi overnight. The catalyst was filtered off and the solvent evaporated. Theresidue (78 g, 98%) was used in the next step without furtherpurification. The free base gave ¹H-NMR (CDC₁ ₃) δ1.00 (t, 3H), 1.55 (m,4H), 2.25 (s, 3H), 2.80 (t, 2H), 3.20 (m, 4H), 6.95 (s, 2H); ¹³C-NMR(CDCl₃): δ12.54, 20.69, 22.53, 24.72, 27.65, 39.92,40.29,44.59, 131.71,133.21, 139.82, 142.09. FAB-MS (m/z) 299 (M⁺+1). Mesitylenesulfonylchloride (8.8 g, 40.5 mmol) in dioxane (30 ml) was added dropwise to astirred mixture of compound 3 (7.8 g, 27 mmol) dissolved in dioxane (60ml) and 50% KOH (30 ml) at 0° C. The reaction mixture was allowed toreach 20° C. and then kept over night. An excess of water was added andthe mixture was extracted with chloroform, dried (Na₂SO₄) andevaporated. The oily residue was crystallized from ethyl acetate/hexaneyielding 4; 10.9 g (82%); mp 71.5-72° C. ¹H-NMR (CDC₁ ₃) δ1.00 (t, 3H),1.10-1.50(m, 4H), 2.30 (s, 6H), 2.55, 2.60 (s, 12H), 2.85 (q, 2H), 3.15(m, 4H), 4.70 (t, 1H), 6.95, 7.00 (s, 4H); ¹³C-NMR (CDCl₃): δ12.70,20.92, 21.04, 22.73, 22.92, 24.58, 26.68, 40.04, 42.02, 44.42, 131.91,133.31, 133.64, 138.99, 140.05, 142.15, 142.35. MS-FAB (m/z) 480 (M⁺).

[0103] (E)-2-Butene-1,4-diyl bis[mesitylenesulfonate](5):(E)-2-Butene-1,4-diol (1.76 g, 20 mmol), and benzyltriethylammoniumbromide (270 mg, 1 mmol) were dissolved in 30 ml of a 50% potassiumhydroxide solution and 30 ml of dioxane. The mixture was stirred at 5°C. and mesitylenesulfonyl chloride (8.72 g, 40 mmol) dissolved in 30 mlof dioxane was added dropwise. When the addition was over, stirring wascontinued for 1 h, water was then added, and the white precipitate wasfiltered and crystallized from chloroform-hexane to yield 5; 7.0 g(77%); mp 119-120° C. ¹H-NMR (CDCl³): δ2.35 (s, 6H), 2.60 (s, 12H), 4.45(d, 41), 5.75 (b, 2H), 6.95 (s, 4H); ¹³C-NMR (CDCl₃): δ20.96, 22.52,67.96, 127.67, 131.69, 131.74, 139.79, 143.45. MS-EI (m/z), 452 (M⁺),253, 200, 183. Anal. Calcd for C₂₂H₂₈O₆S₂: C, 58.40; H, 6.19. Found: C,58.35; H, 6.22.

[0104] Compound 6 was synthesized from 5 according to a proceduredescribed elsewhere (Reddy et al., J. Med. Chem. 41:4723 (1998)) in 56%yield. ¹-NMR (CDCl₃): δ0.95 (t, J=7.15 Hz, 6H, CH₃), 1.34 (m, 8H, CH₂),2.29 (s, 12H, CH₃), 2.55 (s, 24H, CH₃), 3.09 (m, 12H, NCH₂), 3.72 (d,J=4.53 Hz, 4H, NCH₂), 5.48 (t, J=4.31 Hz, 2H, CH═CH), 6.92 (s, 4H, Ph),6.93 (s, 4H, Ph); ¹³C-NMR (CDCl₃): δ12.71, 20.90, 22.71, 22.76, 24.74,40.04, 42.21, 44.56, 45.69, 128.45, 131.88, 132.02, 140.05, 140.16,142.20, 142.58. MS-FAB (m/z) 1012.8 (M⁺, 100%), 828.7, 646.7, 561, 176.

[0105] Compound 7 was obtained from 6 as described elsewhere (Reddy etal., J. Med. Chem. 41:4723 (1998)) in 75% yield, mp >230° C. ¹H-NMR(D₂O): δ1.26 (t, J=12.5 Hz, 6H, 2CH₃), 1.79 (m, 8H, CH₂), 3.12 (m, 12H,NCH₂), 3.80 (d, J=7.16, 4H, NCH₂), 6.10 (m, 2H, CH═CH); ¹³C-NMR (D₂O):δ12.79, 25.10, 45.19, 48.53, 48.62, 50.36, 130.66. MS-MALDI (m/z): 285.3(MH⁺, 100%).

[0106] Compound 8 was obtained from the commercially available butynediol. Mesitylenesulfonyl chloride (19.5 g, 90 mmol) in dioxane (30 ml)was added dropwise to a stirred and cooled mixture of butyne diol (2.58g, 30 mmol), 50% potassium hydroxide (30 ml) and triethylbenzyneammonium bromide (405 mg, 1.5 mmol). Once the addition was over, themixture was stirred at room temperature for an additional 3 h. An excessof water was added and the white precipitate was cooled over night,filtered off and dried. Recrystallization from ethyl acetate/hexaneafforded 8.6 g (63%) of 8; mp 105-106° C. ¹H-NMR (CDCl₃): δ2.30 (s, 6H),2.60 (s, 12H), 4.50 (s, 4H), 6.95 (s, 4H); ¹³C-NMR (CDCl3): δ20.93,22.48, 56.13, 80.41, 130.65, 131.67, 139.98, 143.67. MS-EI (m/z) 450(M⁺).

[0107] Compound 9 was obtained following a procedure analogous to thatdescribed for compound 42 (see below). From 450 mg (1 mmol) of diester 8and 1.05 g (2.2 mmol) of diamide 4, 570 mg (56%) of tetramide 9 wasobtained. ¹H-NMR (CDCl₃): δ0;90 (t, 6H); 1.30 (bs, 8H), 2.20 (s, 12H),2.45 (s, 24H), 3.05 (m, 12H), 3.75 (s, 4H), 6.87 (s, 8H); ¹³C-NMR(CDCl₃): δ12.70, 20.78, 22.68, 34.65, 39.97, 44.46, 44.99, 78.62,131.85, 131.98, 132.34, 140.14, 142.13, 142.55. MS-FAB (m/z) 1010(M^({circle over (+)})).

[0108] Compound 10 was obtained following a procedure analogous to thatdescribed for compound 43 (see below). From 500 mg (0.49 mmol) oftetramide 9, 160 mg (76%) of the tetrahydrochloride6 25 was obtained;mnp >280° C. (decomp). ¹H-NMR (D₂O): δ1.30 (t, 6H), 1.80 (b, 8H),2.90-3.25 (m, 12H), 4.05 (s, 4H); ¹³C-NMR (2°): δ13.39, 25.64, 39.26,45.72, 49.00, 49.20, 81.20. MS-MALDI 283 (M⁺+1).

[0109] Compound 11: Mesitylenesulfonylethylamide 1 (3.1 g, 13.65 mmol)was dissolved in anhydrous DMF (30 ml) followed by the addition of NaH(85%, 0.423 g) in several portions. The mixture was stirred at roomtemperature for 1 h. N-(4-chloro-2-butenyl)-phthalimide (Aldrich, 3.06g, 13 mmol) in 20 ml of DMP was added to the flask followed by stirringat 80° C. over night. The mixture was cooled to room temperature,quenched with H₂O (10 ml), and the solution was evaporated to dryness invacuo. The solid residue was partitioned between 25 ml H₂O and 25 CHCl₃.The aqueous layer was extracted with CHCl₃ (3×25 ml), the organic layerswere washed with brine (35 ml), dried (MgSO₄), the solvent wasevaporated to afford a gum which solidified upon trituration with hexaneto give 11. The ¹H-NMR and ¹³C-NMR spectra showed that 11 was pureenough to be used in the next step without further purification, yield4.75 g. ¹H-NMR (CDCl₃): δ1.16 (t, J=7.11 Hz, 3H, CH₃), 2.29 (s, 3H,CH₃), 2.63 (s, 6H, 2CH₃), 3.29 (q, J=7.11 Hz, 2H, CH₂), 4.06 (d, J=5.24Hz, 2H, NCH₂), 4.26 (d, J=5.72 Hz, 2H, NCH₂), 5.59 (m, 2H, CH═CH), 6.95(s, 2H, Ph), 7.71 (m, 2H, Ph), 7.83 (m, 2H, Ph); ¹³C-NMR (CDCl₃):δ13.06, 20.89, 22.72, 34.35, 40.68, 42.01, 123.27, 126.69, 129.47,131.90, 134.00, 140.24.

[0110] Compound 12: Amide 11 (20 g, 46.95 mmol) was dissolved inmethanol, hydrazine monohydrate (5 ml, 98.52 mmol) was added and thesolution stirred at 55° C. for 24 h. Initially it was a homogeneoussolution; however, after several hours a white solid precipitated. Thenixture was cooled to room temperature, 300 ml of conc. HCl were addedslowly (exothermic reaction), and stirring at room temperature wascontinued for 12 h more. Methanol was evaporated, and the resultingsolid was extracted with CHCl₃ (3×150 ml). The aqueous layer wasneutralized with 50% NaOH, extracted again with CHCl₃ (3×100 ml), thecombined organic layers were dried (MgSO₄); the solution was evaporatedto afford a gum, which solidified under high vacuum to give 12; yield9.0 g (65%). The compound was purified by column chromatography usinghexane, ethyl acetate (7:3) as eluent; mp 167-169° C. ¹H-NMR (CDCl₃):δ1.0 (t, J=7.1 Hz, 3H, CH₃), 2.28 (s, 3H, CH₃), 2.56 (s, 6H, 2CH₃), 2.62(br, NH2), 3.12 (q, J=7.1 Hz, 2H, NCH₂), 3.73 (br, 2H, NCH₂), 3.94 (d,J=6.0 Hz, 2H, NCH₂), 5.80 (m, 2H, CH═CH), 6.92 (s, 2H, Ph); ¹³C-NMR(CDCl₃): δ12.97, 20.93, 22.74, 36.43, 40.94, 42.08, 124.29, 131.89,132.00, 132.62, 140.21, 142.67.

[0111] Compound 13 was obtained from 12 as described for 4 in 96% yield.It was purified 15 by column chromatography using hexane and ethylacetate (4:1.5) as eluants; mp 98-99° C.; ¹H-NMR (CDCl₃): δ0.93 (t,J=5.85 Hz, 3H, CH₃), 2.23 (s, 3H, CH₃), 2.24 (s, 3H, CH₃), 2.50 (s, 6H,2CH₃), 2.56 (s, 6H, 2CH₃), 3.06 (q, J=7.15 Hz, 2H, NCH₂), 3.48 (t,J=5.99 Hz, 2H, NCH₂), 3.68 (d, J=5.72 Hz, 2H, NCH₂), 4.58 (t, J=6.24 Hz,1H, NH), 5.44 (m, 2H, CH═CH), 6.87 (s, 2H, Ph), 6.89 (s, 2H, Ph);¹³C-NMR (CDCl₃): δ12.80, 20.89, 22.64, 22.89, 39.01, 40.59, 41.41,128.14, 128.46, 131.91, 131.96, 139.08, 140.19, 142.26, 142.54. MS-FAB(m/z) 479.2 (M⁺, 65%), 296.2, 279.1, 267.2, 183.1.

[0112] Compound 15: Amide 13 (4.79 g, 10 mmol) was dissolved inanhydrous DMF (40 ml) followed by addition of NaH (0.37 g) in severalportions, the mixture stirred at room temperature for 2 h,cis-1,4dichloro-2-butene (7.5 g, 60 mmol) in 10 ml DMF was added atonce, and stirring was continued at 50° C. over night. The mixture wascooled to room temperature, quenched with 10 ml H₂O, the solvents wereevaporated, and the contents were partitioned between H₂O (50 ml) andCHCl₃ (50 ml). The aqueous layer was extracted with CHCl₃ (3×50 ml), thepooled organic layers were dried (MgSO₄), evaporated, and 15 waspurified by column chromatography using hexane, ethyl acetate (8.5:1.5)as eluants; yield 5.5 g (97%), mp 106-108° C. ¹H-NMR (CDCl₃): δ1.03 (t,J=7.33 Hz, 3H, CH₃), 2.30 (s, 6H, 2CH₃), 2.57 (s, 12H, 4CH₃), 3.17 (q,J=7.31 Hz, NCH₂), 3.71 (m, 4H, NCH₂), 3.81 (d, J=6.87 Hz, 2H, NCH₂),3.95 (d, J=7.70 Hz, 2H, CHCl₂), 5.50 (m, 3H, CH═CH), 5.74 (m, 1H,CH═CH), 6.93 (s, 2H, Ph), 6.95 (s, 2H, Ph); ¹³C-NMR (CDCl₃): δ12.91,22.70, 22.74, 38.20, 40.45, 41.60, 42.11, 42.33, 128.17, 128.95, 129.34,129.40, 131.94, 132.08, 140.23, 140.34, 142.91. MS-FAB (m/z) 566.7 (M⁺,100%), 153.4, 96.3.

[0113] Compound 14 was prepared from 13 and 1,4diiodobutane as describedabove for 15. The product was purified by column chromatography usinghexanes and ethyl acetate (4:1) as eluant; yield 79%. ¹H-NMR (CDCl₃):δ1.04 (t, J=7.10 Hz, 3H, CH₃), 1.63 (m, 4H, CH₂), 2.30 (s, 6H, 2CH₃),2.58 (s, 12H, 4CH₃), 3.04 (t, J=6.50 Hz, 2H, CH₂I), 3.16 (m, 4H, NCH₂),3.78 (d, J=5.14 Hz, 4H, NCH₂), 5.55 (m, 2H, CH═CH), 6.94 (s, 2H, Ph),6.95 (s, 2H, Ph); ¹³C-NMR (CDCl₃): δ5.69, 12.92, 20.95, 22.72, 22.78,28.25, 30.36, 40.47, 41.59, 42.11, 44.71, 128.34, 129.00, 131.94,132.06, 132.60, 132.89, 140.15, 140.21, 142.50, 142.71.

[0114] Compound 16 was prepared from 4 and 4-bromobutyronitrile asdescribed above for Compound 2 in 94% yield. ¹NMR(CDCl₃): δ0.97 (t,J=7.12Hz, 3H, CH₃), 1.40 (m, 4H, 2CH₂), 1.85 (Pent., m, 2H, CH₂), 2.27(t, J=7.17 Hz, 2H CH₂CN), 2.30 (s, 6H, 2CH₃), 2.57 (s, 6H, 2CH₃), 2.58(s, 6H, 2CH₃), 3.13 (m, 6H, NCH₂), 3.28 (t, J=7.11 Hz, 2H, NCH₂), 6.94(s, 2H, Ph), 6.96 (s, 2H, Ph); ¹³C NMR (CDCl₃): δ12.55, 14.54, 20.84,22.64, 22.73, 23.65, 24.43, 24.57, 39.88, 44.31, 44.54, 45.58, 118.69,131.84, 132.05, 132.73, 133.36, 139.94, 142.20, 142.71.

[0115] Compound 17 was prepared from 16 as described above for Compound3 in 93% yield. ¹NMR(CDCl₃): δ1.00 (t, J=6.92Hz, 3H, CH₃), 1.40 (m, 10H,4CH₂, NH₂), 2.29 (s, 6H, 2CH₃), 2.57 (b, 14H, 4CH₃, CH₂N), 3.13 (m, 8H,4CH₂N), 6.93 (s, 4H, 2 Ph); ¹³C NMR (CDCl₃): 12.72, 20.90, 22.72, 22.78,24.67, 24.80, 30.80,40.02, 41.61, 44.56,45.10,45.38, 131.87, 140.04,142.21, 142.28; MS-FAB(M/Z) 552.3(M⁺, 100%), 368.2, 299.1, 183.0, 154.0.

[0116] Compound 18 was prepared from 17 as described above for Compound4. ¹H NMR(CDCl₃): δ0.96 (t, J=7.13Hz, 3H, CH₃), 1.38 (m, 8H, 4CH₂), 2.29(s, 9H, 3CH₃), 2.55 (s, 6H, 2CH₃), 2.56 (s, 6H, 2CH₃); 2.59 (s, 6H,2CH₃), 2.80 (m, 2H, CH₂N), 3.10 (m, 8H, NCH₂), 4.67(t, J=6.6Hz, 1H, NH),6.93 (s, 6H, 3 Ph); ¹³C NMR(CDCl₃): δ12.56, 20.87, 22.70, 22.74, 22.84,24.40, 26.45, 24.67, 26.62, 39.87, 41.88, 44.45, 45.02, 45.09, 131.86,131.90, 131.92, 133.12, 133.32, 133.68, 138.91, 139.97, 142.02, 142.21,142.38; MS-FAB(M/Z): 756.9(M+23(Na), 100%) 572.8, 390.7, 333.6, 305.6

[0117] Compound 19 was prepared from 4 and 1,4-dichloro-2-butene asdescribed above for 15 in 99% yield. ¹H-NMR (CDCl₃): δ1.01 (t, J=7.11Hz, 3H, CH₃), 1.38 (m, 4H, CH₂), 2.29 (s, 3H), 2.30 (s, 3H), 2.57 (s,6H), 2.61 (s, 6H), 3.11 (m, 4H, NCH₂), 3.16 (q, J=7.15 Hz, 2H, NCH₂),3.81 (d, J=7.17 Hz, 2H, NCH₂), 3.98 (d, J=8.05 Hz, 2H, CH₂Cl), 5.51 (m,1H, CH═CH), 5.77 (m, 1H, CH—CH), 6.93 (s, 2H, Ph), 6.95 (s, 2H, Ph);¹³C-NMR (CDCl₃): δ12.76, 20.91, 22.71, 22.76, 24.74, 38.12, 40.08,41.85, 44.59,45.54, 129.14, 129.25, 131.88, 132.02, 140.09, 140.19,142.21, 142.63. MS-FAB (m/z) 569.3 (M⁺, 20%), 385.2, 240.1, 203.3,183.0, 119 (100%).

[0118] Compound 20 was prepared from 18 and 15 following the proceduredescribed above for 15. It was purified by column chromatography usinghexanes-ethyl acetate (7:3) as eluant (78% yield). ¹H-NMR (CDCl₃): δ0.97(t, J=7.10 Hz, 3H, CH₃), 0.99 (t, J=7.0 Hz, 3H, CH₃), 1.29 (m, 8H, CH₂),2.29 (s, 15H, CH₃), 2.54, 2.55, 2.59 (s, 30H, CH₃), 3.06 (m, 12H, NCH₂),3.65 (m, 8H, NCH₂), 5.48 (m, 4H, CH═CH), 6.92 (s, 10H, Ph); ¹³C-NMR(CDCl₃): δ12.70, 12.83, 20.88, 20.91, 22.65, 22.68, 22.72, 22.74, 24.48,24.72, 40.04, 40.47, 41.53, 42.07, 42.22, 42.34, 44.54, 44.96, 127.94,128.27, 128.57, 129.20, 131.92, 132.05, 139.96, 140.00, 140.12, 140.16,140.27, 142.19, 142.25, 142.47, 142.58, 142.87. MS-FAB (m/z) 1263.81(M⁺, 100%), 1080.01, 898.11, 714.81, 563.

[0119] Compound 21: Pentamide 20 (0.93 g, 0.735 mmol) was dissolved in20 ml anhydrous CH₂Cl₂, phenol (3.46 g, 36.77 mmol) was added, followedby HBr in acetic acid (30%, 17.62 ml) and the mixture was stirred overnight at 25° C. Water (10 ml) was added to the flask, the aqueous layerwas separated, the organic layer was extracted with 5 ml H₂O, and thecombined aqueous layers were washed with CH₂Cl₂ (6×15 ml). Water wasevaporated under vacuum to afford a solid which was dissolved in 1 ml 1NNaOH followed by 1 ml of 50% KOH. This solution was extracted with CHCl₃(10×5 ml). The combined organic layers were dried (MgSO₄), CHCl₃ wasevaporated, and the residue dissolved in anhydrous diethyl ether.Anhydrous HCl gas was passed into the solution while cooling at 0° C. Awhite solid precipitated which was filtered and washed with ether. Itwas 21 (84%). ¹H-NMR (D₂O): δ1.29 (t, J=7.32 Hz, 3H, CH₃), 1.31 (t,J=7.24 Hz, 3H, CH₃), 1.79 (m, 8H, CH₂), 3.12 (m, 12H, NCH₂), 3.87 (m,8H, NCH₂), 5.98 (m, 4H, CH═CH); ¹³C-NMR (D₂O): δ13.36, 13.46, 25.66,25.77, 45.44, 45.74, 46.24, 46.41, 46.84, 49.09, 49.41, 49.70, 129.02,129.16, 129.47, 129.66. MS-MALDI (m/z) 354.36 (MH⁺, 100%).

[0120] Compound 22 was prepared in 51% yield from 18 and 14 as describedabove for compound 15. ¹H-NMR (CDCl₃): δ0.97 (t, J=6.59 H, 3H, CH₃),0.99 (t, J=7.02 Hz, 3H, CH₃), 1.29 (m, 12H, CH₂), 2.29 (s, 15H, CH₃),2.55 (s), 2.56 (s), 2.57 (s), 3.10 (m, 16H, NCH₂), 3.70 (m, 4H, NCH₂),5.47 (m, 2H, CH═CH), 6.93 (s, 10H, Ph); ¹³C-NMR (CDCl₃): δ12.69, 12.83,20.91, 22.69, 22.71, 22.76, 24.43, 24.70, 40.48, 41.11, 41.48, 44.50,44.91, 128.13, 128.90, 131.88, 131.94, 132.01, 133.29, 139.95, 140.00,140.15, 142.22, 142.29, 142.60. MS-FAB (m/z) 1265.91 (M⁺, 100%),1082.01, 900.11, 716.91, 563.81.

[0121] Compound 23 was prepared from 22 in 79% yield as described abovefor 21. ¹H-NMR (D₂O): δ1.29 (t, J=7.29 Hz, 3H, CH₃), 1.30 (t, J=7.30 Hz,3H, CH₃), 1.78 (m, 12H, CH₂), 3.12 (m, 16H, NCH₂), 3.83 (m, 4H, NCH₂),5.96 (m, 2H, CH═CH); ¹³C-NMR (D₂O): δ13.31, 13.42, 25.62, 25.75, 45.38,45.71, 46.18, 46.76, 49.07, 49.32, 49.69, 129.11, 129.39. MS-MALDI (m/z)356.38 (MH⁺, 100%).

[0122] Compound 24 was prepared from 18 (52% yield) as described. ¹H-NMR(CDC₁ ₃): δ0.95 (m, 6H, 2CH₃), 1.32 (m, 12H, CH₂), 2.29 (s, 15H, CH₃),2.55 (s, 30H, CH₃), 3.06 (m, 16H, NCH₂), 3.70 (m, 4H, NCH₂), 5.47 (m,2H, CH═CH), 6.92 (s, 10H, Ph); ¹³C-NMR (CDCl₃): δ12.67, 20.90, 22.71,22.76, 24.43, 24.68, 39.97, 42.08, 44.48, 44.90, 45.61, 128.28, 128.45,131.87, 131.93, 132.01, 139.96, 140.00, 140.12, 142.21, 142.28, 142.58.MS-FAB (m/z) 1265.91 (M⁺, 100%), 1082.01, 900.11.

[0123] Compound 25 was prepared from 24 in 96% yield as described abovefor 21. ¹H-NMR (D₂O): δ1.28 (t, J=7.29 Hz, 6H, 2CH₃), 1.78 (m, 12H,CH₂), 3.09 (m, 16H, NCH₂), 3.84 (m, 4H, NCH₂), 5.96 (m, 2H, CH═CH);¹³C-NMR (D₂O): δ13.31, 25.61, 25.73, 45.70, 46.79, 49.05, 49.36, 49.65,129.19. MS-MALDI (m/z) 356.4 (MH⁺).

[0124] Compound 26: A mixture of KOH (0.25 g), K₂CO₃ (0.25 g) andtetra-n-butyl-ammonium hydrogen bromide (0.05 g) were suspended in 15 mlbenzene. Mesitylenesulfonylamide (0.199 g, 1 mmol) was added to thesuspension and the mixture was heated to 50° C. Iodide 14 (1.98 g, 3mmol) in 10 ml benzene was added to the flask, the mixture heated underreflux over night, then cooled to room temperature; the inorganic solidswere filtered off and washed with benzene (2×20 ml). The combinedorganic layers were washed several times with water until the washingswere neutral. The benzene was dried (MgSO₄), evaporated and the residuepurified by column chromatography using hexanes and ethyl acetate(7.5:2.5) as eluant; 25% yield (0.948 g). ¹H-NMR (CDCl₃): δ1.00 (t,J=7.18 Hz, 6H, CH₃), 1.28 (m, 8H, CH₂), 2.29 (s, 15H, CH₃), 2.53 (s),2.55 (s), 2.57 (s), 3.03 (m, 8H, NCH₂), 3.12 (q, J=7.13 Hz, 4H, NCH₂),3.70 (m, 8H, NCH₂), 5.47 (m, 4H, CH═CH), 6.93 (s, 10H, Ph); 13C-NMR(CDCl₃): δ12.78, 20.85, 22.63, 22.69, 24.32, 24.58, 40.41, 41.43, 42.00,44.76, 45.43, 128.08, 128.83, 131.88, 131.95, 132.77, 132.85, 133.23,139.90, 140.04, 140.08, 142.22, 142.43, 142.53. MS-FAB (m/z) 1263.81(M⁺, 100%), 1081, 898.11, 815.01, 561.81, 418.81.

[0125] Compound 27 was prepared from 26 in 57% yield as described abovefor 21. 1H-NMR (D₂O): δ1.31 (t, J=7.31 Hz, 6H, CH₃), 1.78 (m, 8H, CH₂),3.15 (m, 12H, NCH₂), 3.83 (m, 8H, NCH₂), 5.96 (m, 4H, CH═CH); ¹³C-NMR(CDCl₃): δ13.43, 25.64, 25.76, 45.39, 46.19, 46.77, 49.35, 49.72,129.11, 129.41. MS-MALDI (m/z) 354.3 (MH⁺, 100%).

[0126] Compound 28 was prepared from 15 and mesitylenesulfonylamide in24% yield as described above for 26; mp 57.7° C. ¹H-NMR (CDCl₃): δ0.99(t, J=7.09 Hz, 6H, CH₃), 2.29 (s, 15H, CH₃), 2.53 (s), 2.55 (s), 3.12(q, J=7.09 Hz, 4H, NCH₂), 3.63 (m, 16H, NCH₂), 5.49 (m, 8H, CH═CH), 6.93(s, 10H, Ph); ¹³C-NMR (CDCl₃): δ12.85, 20.89, 20.92, 22.66, 40.47,41.53, 42.19, 128.00, 128.47, 128.58, 129.11, 131.92, 132.05, 140.17,140.30, 142.46, 142.87. MS-FAB (m/z) 1259.81 (M⁺, 60%), 1075.91, 894.01,306.51, 153.4 (100%).

[0127] Compound 29 was prepared from 28 in 81% yield as described abovefor 21. ¹H-NMR (D₂O): δ1.31 (t, J=7.29 Hz, 6H, CH₃), 3.15 (q, J=7.31 Hz,4H, NCH₂), 3.84 (m, 4H, NCH₂), 3.90 (m, 12H, NCH₂), 5.98 (m, 8H, CH═CH);¹³C-NMR (D₂O): δ13.42, 45.41, 46.22, 46.44, 129.07, 129.37, 129.42,129.58. MS-MALDI (m/z) 350.31 (MH⁺).

[0128] Compound 30 was prepared from 19 in 25% yield as described abovefor 26; mp 62.3° C. ¹H-NMR (CDCl₃): δ0.95 (5, J=7.17 Hz, 6H, CH₃), 1.33(m, 8H, CH₂), 2.29 (s, 15H, CH₃), 2.54 (s), 2.55 (s), 3.07 (m, 12H,NCH₂), 3.65 (m, 8H, NCH₂), 5.48 (m, 4H, CH═CH), 6.92 (s, 10H, Ph);¹³C-NMR (CDCl₃): δ12.69, 20.90, 22.69, 22.73, 24.70, 40.03, 42.13,42.30, 44.53, 45.59, 128.11, 128.79, 131.87, 132.00, 140.02, 140.14,140.28, 142.17, 142.58, 142.85. MS-FAB (m/z) 1263.81 (M⁺, 100%),1080.01, 898.11, 714.01, 153.

[0129] Compound 31 was prepared from 30 in 87% yield as described abovefor 21. ¹H-NMR (D₂O): δ1.28 (t, J=7.32 Hz, 6H, CH₃), 1.79 (m, 8H, CH₂),3.10 (m, 12H, NCH₂), 3.87 (m, 8H, NCH₂), 5.98 (m, 4H, CH═CH), ¹³C-NMR(D₂O): δ12.70, 25.00, 25.13, 45.10, 45.81, 46.21, 48.44, 48.78, 128.44,128.85. MS-MALDI (m/z) 354.3 (MH⁺).

[0130] Compound 32: Mesitylenesulfonylamide (1.47 g, 7.38 rnmol) wasdissolved in 50 ml anhydrous DMF, and NaH (85%, 0.3 g) was added to itunder a nitrogen atmosphere. The mixture was stirred at room temperatureand 19 ( 1.40 g, 2.46 mmol) in 25 ml DMP were added. Heating at 65° C.continued over night. The mixture was cooled to room temperature, and 10ml of H₂O were added. The solvents were evaporated and the solid residuewas partitioned between 40 ml H₂O and 40 ml CHCl₃. The aqueous layer wasextracted with CHCl₃ (2×30 ml), the combined organic layers were washedwith H₂O (3×50 ml), dried (MgSO₄), and evaporated. The residue waspurified by column chromatography using hexanes-ethyl acetate (7.5:2.5).1.7 g (97%) of 32 as a white solid was obtained. ¹H-NMR (CDCl₃): δ0.94(t, J=7.10 Hz, 3H, CH₃), 1.30 (m, 4H, CH₂), 2.29 (s), 2.30 (s), 2.55 (s,12H, CH₃), 2.65 (s, 6H, CH₃), 3.11 (m, 6H, NCH₂), 3.52 (m, 1H, NCH),3.65 (m, 2H, NCH₂), 3.71 (m, 1H, NCH₂), 4.82 (br, 1H, NH), 5.47 (m, 2H,CH═CH), 6.93 (s, 4H, Ph), 6.96 (s, 2H, Ph); ¹³C-NMR (CDCl₃): δ12.50,20.91, 22.71, 22.76, 22.83, 22.91, 24.66, 38.98, 39.85, 42.15, 42.26,44.50, 128.06, 128.51, 131.86, 131.91, 138.18, 140.00, 140.14, 140.28,142.17, 142.65.

[0131] Compound 33 was prepared from 32 and 14 in 51% yield as describedabove for 22. ¹H-NMR (CDCl₃): δ0.99 (5, J=7.19 Hz, 6H, CH₃), 1.33 (m,8H, CH₂), 2.29 (s, 15H, CH₃), 2.55 (s), 2.57 (s), 3.10 (m, 12H, NCH₂),3.70 (m, 4H, NCH₂), 3.77 (m, 4H, NCH₂), 5.42 (m, 4H, CH═CH), 6.93 (s,10H, Ph); ¹³C-NMR (CDCl₃): δ12.70, 12.71, 20.89, 22.66, 22.72, 22.78,22.81, 24.60, 26.53, 40.39, 41.37, 41.87, 42.20, 45.47, 128.26, 128.62,131.78, 131.84, 131.86, 131.92, 132.77, 138.92, 139.96, 140.09, 140.17,142.57, 142.63.

[0132] Compound 34 was prepared from 33 as described above for 21 in 40%yield.

[0133] Compound 35 was prepared from 15 in 94% yield as described abovefor 32.

[0134] Compound 36 was prepared from 35 and 14 in 82% yield as describedabove for 33. ¹H-NMR (CDCl₃): δ0.99 (t, J=7.11 Hz, 6H, CH₃), 1.33 (m,4H, CH₂), 2.29 (s, 15H, CH₃), 2.55 (s), 2.57 (s), 3.07 (m, 8H, NCH₂),3.70 (m, 12H, NCH₂), 5.46 (m, 6H, CH═CH), 6.92 (s, 10H, Ph); ¹³C-NMR(CDCl₃): δ12.69, 12.80, 20.84, 22.62, 22.68, 22.73, 22.77, 24.58, 26.55,40.44, 41.51, 41.86, 42.04, 42.24, 45.49, 128.10, 128.25, 128.52,128.62, 128.82, 131.89, 131.95, 132.79, 138.89, 140.07, 140.14, 140.23,141.94, 142.44, 142.53, 142.82. MS-FAB (m/z) 1262.8 (M⁺, 75%), 1080.01,896, 119 (100%).

[0135] Compound 37 was prepared from 36 in 65% yield as described abovefor 21. ¹H-NMR (D₂O): δ1.31 (t, J=6.97 Hz, 6H, CH₃), 1.79 (m, 4H, CH₂),3.12 (m, 8H, NCH₂), 3.83 (m, 12H, NCH₂), 5.96 (m, 6H, CH═CH); ¹³C-NMR(D₂O): δ13.48, 25.69, 26.76, 41.67, 45.44, 46.24, 46.45, 46.80, 49.41,129.00, 129.12, 129.45, 129.71. MS-MALDI (m/z) 352.3 (M⁺).

[0136] Compound 38 was prepared from 35 and 19 in 89% yield asdescribed. ¹H-NMR (CDCl₃): δ0.95 (m, 6H, CH₃), 1.33 (m, 4H, CH₂), 2.29(s, 15H, CH₃), 2.54 (s), 2.55 (s), 2.57 (s), 3.09 (m, 8H, NCH₂), 3.66(m, 12H, NCH₂), 5.48 (m, 6H, CH═CH), 6.93 (s, 10H, Ph); ¹³C-NMR (CDCl₃):δ12.51, 12.63, 20.84, 20.86, 22.63, 22.65, 22.84, 24.61, 38.92, 40.40,41.40, 42.11, 42.18, 44.44, 45.48, 127.95, 128.07, 128.49, 128.62,128.80, 131.76, 131.83, 131.85, 131.88, 132.01, 138.05, 139.01, 140.07,140.13, 140.24, 142.15, 142.21, 142.87. MS-FAB (m/z) 1263.1 (M⁺, 90%),1080.1, 896.01, 119 (100%).

[0137] Compound 39 was prepared from 38 in 54% yield as described abovefor 21; mp 270° C. (dec.). ¹H-NMR (D₂O): 8-1.31 (m, 6H, CH₃), 1.80 (m,4H, CH₂), 3.10 (m, 8H, NCH₂), 3.86 (m, 12H, NCH₂), 5.98 (m, 6H, CH═CH);¹³C-NMR (D₂O): δ13.30, 13.42, 25.58, 25.70, 45.69, 46.21, 46.43, 46.81,49.02, 49.37, 129.00, 129.15, 129.37, 129.59. MS-MALDI (m/z): 352.343(MH⁺).

[0138] Compound 42: NaH (80%, 132 mg, 4.4 mmol) was added to a solutionof diamide 41 (1.98 g, 4.4 mmol) in DMF (10 ml). The mixture was stirredat 20° C. for 30 minutes and a solution of the diester 40 (Reddy et al.(1998) J. Med Chem., 41:4723) (960 mg, 2 mmol) in DMF (10 ml) was addeddropwise. The mixture was stirred at 75° C. for 2 h, the solvent wasdistilled off, the residue was taken in chloroform, washed with asaturated solution of ammonium chloride, dried (Na₂SO₄) and evaporatedto dryness. The crude oil was purified by column chromatography usinghexane-ethyl acetate (8:2) as running solvent. 1.4 g (70%) was obtainedas a glassy oil. ¹³C-NMR (CDCl₃): δ20.58, 22.63, 22.80, 32.42,33.86,43.16,45.42, 46.26, 132.75, 133.21, 139.82, 142.40. MS-FAB 984(M⁺),

[0139] Compound 43: Phenol (1.86 g, 19,7 mmol) and 30% HBr in glacialacetic acid (35 ml) were added in that order to a solution of 42 (600mg, 0.6 mmol) in CH₂Cl₂ (35 ml) at room temperature. The solution wasstirred for 24 h, water (30 ml) was added, followed by extraction withmethylene chloride (3×20 ml). The aqueous layer was evaporated underreduced pressure and the residue was taken up in 2N NaOH (2 ml) and then50% KOH (2 ml) followed by extraction with chloroform ( 6×10 ml). Afterremoval of chloroform, the residue was taken up in ethanol (15 ml) andacidified with concentrated hydrochloric acid (0.4 ml). The product 43(230 mg, 93%) was recrystallized from aqueous ethanol; mp >270° C.(decomp). ¹H-NMR (D₂O): δ1.95 (m, 2H), 2.05-2.25 (m, 6H), 2.75 (s, 6H),2.90 (b, 2H), 3.10-3.35 (m, 12H); ¹³C-NMR (D₂O): 625.21, 25.24, 35.60,35.64, 47.41, 48.58, 50.87. MS-MALDI (mtz) 240 (M++1).

[0140] Compound 47: NaH (80%, 132 mg, 4.4 mmol) was added to a solutionof diarnide 46 (1.98 g, 4.4 mmol) in DMF (10 ml). The mixture wasstirred at 20° C. for 30 min and a solution of the diester 8 (900 mg, 2mmol) in DMF ( 10 ml) was added dropwise. The mixture was stirred at 75°C. for 2 h. The solvent was distilled off, the residue was taken up inchloroform, washed with a saturated solution of ammonium chloride, dried(NaSO₄) and evaporated to dryness. The oily residue was crystallizedfrom ethyl acetate/hexane 1.2 g (61%); mp 165-166° C. ¹H-NMR (CDCl₃):δ1.08 (t, 3H), 1.75 (m 4H), 2.28 (s, 12H), 2.55 (bs, 24H), 3.10 (m,12H), 3.98 (s, 4H), 6.95 (m, 8H); ¹³C-NMR (CDCl₃): δ12.70, 20.86, 22.64,25.14, 34.85, 40.22, 42.62, 43.37, 78.80, 131.99, 132.26, 133.21,140.26, 142.28, 142.71. MS-FAB (m/z) 982 (M⁺).

[0141] Compound 48 was obtained as described for 47. From 1.2 g (1.22mmol) of tetramide 47, 420 mg (86%) of the tetrahydrochloride 48 wasobtained; mp >270° C. (decomp). ¹H-NMR (D₂O): δ1.29 (t, 6H), 2.13 (m,4H), 3.14 (m, 12H), 4.06 (s, 4H); ¹³C-NMR (D₂O): δ13.34, 25.52, 39.45,45.90, 45.64, 46.71, 81.32. MS-MALDI (m/z) 255 (M⁺+1).

[0142] Compound 44 was obtained as described for 47. From 450 mg (1mmol) of diester 8 and 994 mg (2.2 mmol) of diamide 41, 500 mg (52%) ofthe tetramide 44 was obtained and crystallized from ethylacetate-hexane; mp 155-156° C.

[0143] Compound 45 was obtained as described for 43. From 500 mg (0.52mmol) of tetramide 44, 160 mg (82%) of the tetrahydrochloride 45 wasobtained; mp >270° C. (decomp). ¹H-NMR (D₂O): δ2.15 (m, 4H), 2.73 (s,3H), 3.05-3.40 (m, 8H), 4.10 (s, 4H); ¹³C-NMR (D₂O): δ25.59, 35.66,45.90, 46.57, 48.61.

[0144] Compound 51 is a mixture of cis/trans-isomers. ¹H-NMR (D₂O):δ1.15-2.10 (m, 7H), 2.90 (q, 1H), 3.30-3.80 (b, 2H); ¹³C-NMR (D₂O):δ24.16, 24.97, 28.44, 30.42, 36.58, 37.14, 48.24, 52.27, 55.19, 57.45,64.55, 67.26.

[0145] Compound 52: Mesitylenesulfonylchloride (6.5 g, 30 mmol) indioxane ( 10 ml) was added dropwise to a stirred and cooled mixture ofamine alcohol 51 (1.15 g, 10 mmol), triethylbenzyl ammonium bromide (135mg, 0.5 mmol), 50% KOH (10 ml) and dioxane (10 ml). The reaction mixturewas left over night at 20° C. with magnetic stirring. An excess of waterwas added, the solution was extracted with chloroform (3×30 ml), dried(Na₂SO₄) and evaporated to dryness. The oily residue was chromatographedon a silica-gel column using hexane:ethyl acetate (8:2) as eluants.Crystallization from ethyl acetate-hexane afforded 1.2 g (25%) of pure52; mp 167-168° C. ¹H-NMR (CDCl₃): δ1.35-1.90 (6H), 1.90-2.15 (m, 1H),2.30,2.35 (s, 6H), 2.65 (s, 12H), 3.20 (m, 1H), 3.70 (m, 1H), 3.90 (m,1H), 5.15 (d, 1H), 6.90, 7.00 (s, 4H); ¹³C-NMR (CDCl₃): δ20.73, 20.85,22.15, 22.37, 22.70, 26.94, 32.75, 45.34, 56.09, 70.38, 130.22, 131.57,133.98, 138.68, 139.64, 142.02, 143.10. MS-EI (m/z) 479 (M⁺), 280(M^({circle over (+)})-199).

[0146] Compound 54: NaH (105 mg, 3.5 mmol) was added to a solution ofcompound 52 (1.7 g, 3.5 mmol) in DMF (10 ml). The mixture was stirred at20° C. for 30 min and a solution of compound 53 (1.34 g, 3.85 mmol) inDMF (5 ml) was added in small portions. The mixture was stirred at 75°C. for 2 h. The solvent was distilled off, the residue was taken up inchloroform, washed with a saturated solution of ammonium chloride, dried(Na₂SO₄) and evaporated. The oily residue was purified by columnchromatography (hexane-ethyl acetate 8:2) which gave compound 54 (1.22g, 47%). ¹H-NMR (CDCl₃): δ1.98 (t, 3H), 1.20-2.05 (9H), 2.20 (s, 6H),2.55, 2.65 (s, 12H), 2.70-3.55 (9H), 6.85 (s, 4H); ¹³C-NMR (CDCl₃):δ12.49, 20.80, 21.64, 21.87, 22.88, 28.72, 33.16, 36.13, 39.96, 43.80,47.95, 57.77, 61.26, 131.83, 132.94, 133.14, 138.82, 139.90, 142.07,142.63. MS-FAB (m/z) 628 (M⁺+1), 546 (M⁺-81).

[0147] Compound 55 was obtained following the procedure described forcompound 42. From 1.22 g (1.6 mmol) of bromoderivative 54 and 820 mg(1.76 mmol) of diamide 46, 1.26 g (77%) of tetramide 55 was obtained asa glassy oil. ¹H-NMR (CDCl₃): δ0.80 (t, 6H), 1.20-1.75 (6H), 1.90 (m,1H), 2.15 (s, 12H), 2.35-2.60 (s, 24H), 2.65-3.40 (15H), 6.85 (b, 8H);¹³C-NMR (CDCl₃): δ12.38, 20.71, 22.52, 22.66, 24.72, 27.55, 28.04,39.19, 39.71, 41.02, 42.33, 42.62, 43.37, 48.81, 61.44, 131.76, 131.88,133.10, 133.89, 138.66, 139.93, 142.17, 142.33, 142.57. MS-FAB (m/z)1012 (M⁺), 828 (M⁺-184).

[0148] Compound 56 was obtained following the procedure described forcompound 43. From 1.26 g (1.24 mmol) of tetramide 55, 300 mg (56%) ofthe tetrahydrochloride 56 was obtained; mp >270° C. (decomp). ¹H-NM(D₂O): δ1.35 (t, 6H), 1.60 (m, 1H), 1.80 (b, 3H), 2.15 (b, 6H), 2.50 (b,1H), 3.20 (m, 13H), 3.45 (m, 2H); ¹³C-NMR (D₂O): δ13.23, 25.48, 25.73,25.79, 31.69, 31.99, 43.40, 45.91, 46.43, 46.71, 48.07, 53.20, 75.28.MS-MALDI (m/z) 285 (M⁺+1).

[0149] Compound 57: NaH (80%, 150 mg, 5 mmol) and NaBr (2.5 g, 25 mmol)were added to a solution of compound 52 (2.35 g, 4.9 mmol) in DMF (15ml). The mixture was stirred at 20° C. for 30 min and a solution of1-bromoethane (2.2 g, 25 mmol) in DMF (10 ml) was added in smallportions. The mixture was stirred at 90° C. for 3 h. The solvent wasdistilled off, the residue taken up in chloroform, washed with asaturated solution of ammonium chloride, dried (Na₂SO₄) and evaporated.The product was purified by silica gel chromatography (hexane/ethylacetate 9:1). The oily residue (1.5 g, 79%) crystallized on standing; mp68-69° C. ¹H-NMR (CDCl₃): δ1.10 (t, 3H), 1.30-2.10 (6H), 2.25 (b, 4H),2.60 (s, 6H), 3.20 (m, 2H), 3.35 (m, 2H), 3.60 (m, 2H), 6.95 (s, 2H);¹³C-NMR (CDCl₃): δ16.35, 20.93, 21.79, 22.89, 29.32, 29.37, 36.54,38.12, 44.13, 61.40, 131.99, 132.80, 140.20, 142.52. MS-FAB 389 (M⁺+1),308 (M⁺-80).

[0150] Compound 59 was obtained following the procedure described forcompound 42. From 700 mg (1.80 mmol) of compound 57 and 394 mg (0.9mmol) of diamide 58, 400 mg (37%) of tetrarnide 59 were obtained. ¹H-NMR(CDCl₃): δ0.90 (t,6H), 1.25-1.80 (m,8H), 1.80-2.10 (m,8H), 2.15 (s,12H), 2.40, 2.50 (s, 24H), 2.60-3.35 (m,6H), 2.85, 2.90 (s, 8H); ¹³C-NMR(CDCl₃): δ16.14, 20.85, 21.95, 21.99, 22.55, 25.49, 28.78, 28.88, 31.49,37.87, 40.50, 40.83, 43.85, 44.06, 49.30, 61.42, 131.86, 131.96, 133.09,133.40, 139.93, 139.98, 142.27, 142.40. MS-FAB (m/z) 1052(M^({circle over (+)})), 891 (M⁺-184).

[0151] Compound 60 was obtained following the procedure described forcompound 43. From 400 mg (0.38 mmol) of tetramide 59, 95 mg (53%) of thetetrahydrochloride derivative were obtained; mp >270° C. (decomp.)¹H-NMR (D₂O): δ1.30 (t, 6H), 1.60 (m, 2H), 1.80 (m, 6H), 1.95-2.35 (6H),2.45 (m, 2H), 3.20 (m, 10H), 3.40 (m, 4H); ¹³C-NMR (D₂O): δ13.59, 25.34,25.71, 31.75, 32.00, 43.34, 44.83, 48.02, 53.24, 64.52. MS-MALDI (m/z)325 (M⁺1).

[0152] Compound 62: Mesitylenesulfonylchloride (3.27 g, 15 mmol) indioxane (20 ml) was added dropwise to a stirred solution of 61 (1.3 g,10 rnmol) in dioxane (20 ml) and 50% KOH (15 ml) at 0° C. When additionwas completed, the mixture was left over night at 20° C. Excess waterwas added, the solution cooled and the precipitate filtered off.Crystallization from ethylacetate-hexane gave compound 62 (2 g, 80%); mp115-116° C. ¹H-NMR (CDCl₃): δ2.35 (s, 3H), 2.55 (t, 2H), 2.65 (s, 6H),3.25 (q, 2H), 5.15 (t, 1H), 7.0 (s, 2H); ¹³C-NMR (CDCl₃): δ19.07, 20.82,22.78, 38.37, 117.56, 132.07, 133.0, 138.99, 142.67. MS-EI (m/z) 252(M⁺).

[0153] Compound 63: NaH (80%, 330 mg, 11 mmol) was added to a solutionof compound 62 (2.52 g, 10 mmol) in DMF (20 ml) under N₂. The mixturewas stirred for 30 min and a solution of compound 53 (3.82 g, 11 mmol)in DMF (10 ml) was added in small portions. The mixture was stirred at70° C. for 2h. The solvent was distilled off, the residue taken up inchloroform, washed with a saturated solution of ammonium chloride, dried(Na₂SO₄) and evaporated to dryness. The product was purified bysilica-gel chromatography(hexane-ethyl acetate 8:2). The oily residue(3.0 g, 57%) crystallized on standing; mp 105-106° C. ¹H-NMR (CDCl₃):δ1.00 (t, 3H), 1.75 (m, 2H), 2.35 (s, 6H), 2.60 (14H), 3.10 (m, 6H),3.45 (t, 3H), 6.90, 6.95 (s, 4H); ¹³C-NMR (CDCl₃): δ12.63, 16.94, 20.89,22.67, 25.73, 40.27, 42.19, 42.51, 44.72, 117.36, 131.95, 132.22,140.06, 140.34, 142.52, 143.33. MS-EI (m/z) 519 (M⁺), 429 (M⁺-HCN).

[0154] Compound 65: The nitrile 63 (3.0 g, 5.7 mmol) was dissolved in amixture of ethanol (150 ml) and concentrated hydrochloric acid (1.5 ml).PtO₂ was added (300 mg), the mixture was hydrogenated at 50 psi overnight, the catalyst was filtered off and the solvent evaporated toafford an oily residue of compound 64, which was used in the next stepwithout further purification. Free base ¹H-NMR (CDCl₃): δ1.00 (t, 3H),1.55 (m, 2H), 1.75 (m, 2H), 2.30 (s, 6H), 2.55 (14 H), 2.90-3.30 (8H),6.95 (s, 4H); ¹³C-NMR (CDCl₃): δ12.64, 20.87, 22.69, 25.35, 30.93,39.04, 40.12, 42.65, 43.11, 131.86, 133.10, 140.04, 142.43. MS-FAB (m/z)524 (M⁺+1). Mesitylenesulfonylchloride (1.86 g, 8.55 mmol) in dioxane(15 ml) was added dropwise to a stirred mixture of 64 (3.0 g, 5.7 mmol)dissolved in dioxane (30 ml) and 50% KOH (15 ml) at 0° C. The reactionmixture was allowed to reach room temperature and was kept for further 2h. An excess of water was added and the mixture was extracted withchloroform, dried (Na₂SO₄) and evaporated to dryness. Purification wasachieved by silica gel column chromatography using hexane-ethyl acetate(8:2) as eluant; 2.79 g (69%) of 65 were obtained. ¹H-NMR CDCl₃): δ0.95(t, 3H), 1.60 (m, 4H), 2.30 (s, 9H), 2.50 (s, 12H), 2.65 (s, 6H), 2.85(m, 2H), 3.05 (6H), 3.20 (t, 2H), 5.00 (t, 1H), 6.95 (6H); ¹³C-NMRCDCl₃): δ12.45, 20.81, 22.73, 25.23, 27.46, 39.19, 33.99, 42.49, 42.92,43.17, 131.84, 133.05, 133.82, 138.80, 139.90, 141.92, 142.36, 142.64.MS-FAB (m/z) 705 (M^({circle over (+)})).

[0155] Compound 66 was obtained following the procedure described forcompound 42. From 705 mg (1 mmol) of 65 and 426 mg (1.1 mmol) of 57,470mg (46%) of tetramide 66 was obtained as a glassy product. ¹H-NMRCDCl₃): δ0.85-1.10 (t, 6H), 1.35-2.10 (m, 11H), 2.30 (s, 12H), 2.40-2.65(m, 24H), 2.75-3.55 (m, 13H), 6.95 (m, 8H); ¹³C-NMR (CDCl₃): δ12.64,16.11, 20.91, 22.08, 22.75, 24.81, 25.09, 28.83, 29.07, 37.93, 40.08,40.84, 42.50, 42.81, 43.11, 43.42, 49.11, 61.43. MS-FAB (m/z) 1013(M++1).

[0156] Compound 67 was obtained following the procedure described forcompound 43. From 470 mg (0.46 mmol) of tetramide 66, 142 mg (71%) ofthe tetrahydrochloride derivative was obtained; mp >250° C. (decomp).¹H-NMR (D₂O): δ1.30 (t, 6H), 1.60 (m, 1H), 1.85 (b,s, 3H), 2.15 (m, 6H),2.45 (m, 1H), 3.15 (m, 13H), 3.45 (m, 2H); ¹³C-NMR (D₂O): δ13.29, 13.57,25.34, 25.44, 25.64, 31.68, 31.94, 43.27, 44.80, 45.86, 46.62, 47.42,47.97, 53.19, 64.50. MS-MALDI 285 (M⁺+1), 286 (M⁺+2).

[0157] Compound 68a: 4-Cyanobenzaldehyde (Aldrich, 1.31 g, 10 mmol) wasdissolved in 30 ml anhydrous MeOH followed by the addition of MgSO₄(anhydrous, 1.5 g) and 1,4-diaminobutane (Aldrich, 0.44 g, 5 mmol) andthe mixture was stirred under argon over night. The suspension wascooled in an ice bath and NaBH (2.0 g) was added in portions andstirring continued for 2 h at 0° C. The methanol was evaporated undervacuum and the resulting solid was partitioned between 35 ml H₂O and 50ml CHCl₃. Some of the solid was not soluble in either the H₂ or theCHCl₃ and was filtered off and the aqueous layer was extracted withCHCl₃ (2×25 ml). The pooled organic layers were dried (MgSO₄),evaporated and the solid was recrystallized from ethyl acetate-hexane,yield 1.1 g (35%); mp 90.6-90.8° C. ¹H-NMR (CDCl₃): δ1.43 (broad, 2H,NH), 1.55 (m, 4H, CH₂), 2.63 (m, 4H, NCH₂), 3.85 (s, 4H, benzylic CH₂),7.44 (m, 4H, Ph), 7.60 (m, 4H, Ph); ¹³C-NMR CDCl₃): 827.78, 49.28,53.44, 110.65, 118.88, 128.52, 132.12, 146.21. MS (m/z) 318 (M⁺), 185,145, 131, 116 (100%), 70.

[0158] Compound 68b was prepared from 4-cyano-benzaldehyde and1,5-diaminopentane as described above for 68a; 42% yield; mp 92.9-93.0°C. ¹H-NMR (CDCl₃): δ1.40 (m, 4H, NH, CH₂), 1.50 (m, 4H, CH₂), 2.59 (m,4H, NCH₂), 3.83 (s, 4H, benzylic CH₂), 7.45 (m, 4H, Ph), 7.59 (m, 4H,Ph); ¹³C-NMR (CDCl₃): δ24.86, 29.87, 49.29, 53.40, 110.50, 118.85,128.48, 132.04, 146.19. MS (m/z) 332 (M⁺), 216, 199, 145, 116 (100%),84.

[0159] Compound 68c was prepared from 4-cyanobenzyldehyde and1,6-diaminohexane as described above for 68a; 45% yield; mp 95.6-95.8°C. ¹H-NMR (CDCl₃): δ1.35 (m, 4H, CH₂), 1.50 (m, 6H, NH, CH₂), 2.60 (t,J=6.92 Hz, 4H, NCH₂), 3.84 (s, 4H, benzylic CH2), 7.44 (m, 4H, Ph), 7.60(m, 4H, Ph); ¹³C-NMR (CDCl₃): δ27.17, 30.02, 49.42, 53.50, 110.65,118.92, 128.55, 132.14, 146.27. MS (m/z) 346 (M⁺), 230, 213, 145, 116(100%) 98.

[0160] Compound 69a: Dinitrile 68a (0.75 g, 2.36 mmol) was dissolved inanhydrous THF, lithium bis(trimethylsilyl)amide (9.43 ml of a 1 msolution in THF) was added slowly under argon atmosphere. The mixturewas stirred at room temperature for 2 h; then cooled in an ice bath,followed by the addition of 4 equivalents of 6N HCl in ether. A whitesolid precipitated immediately and was filtered after 12 h. The solidwas recrystallized from ethanol-ether to afford 1.19 g of compound 69a(93%). ¹H-NMR (D₂O): δ1.87 (m, 4H, CH₂), 3.22 (m, 4H, CH₂N), 4.40 (s,4H, benzylic CH₂), 7.74 (m, 4H, Ph), 7.91 (m, 4H, Ph); ¹³C-NMR(DMSO-d6): δ22.68, 46.09, 49.28, 128.10, 128.47, 130.69, 138.15, 165.44.MS-ESI (m/z) 353.2 (M⁺), 177.2 (100%).

[0161] Compound 69b was prepared from 68b in 92% yield as describedabove for 69a. ¹H-NMR (D₂O): δ1.52 (m, 2H, CH₂), 1.80 (m, 4H, CH₂), 3.19(m, 4H, NCH₂), 4.40 (s, 4H, benzylic CH₂), 7.75 (m, 4H, Ph), 7.91 (m,4H, Ph); ¹³C-NMR (DMSO-d6): δ24.90, 26.91, 48.96, 51.88, 130.29, 130.46,132.43, 139.51, 167.52. MS-ESI (m/z) 367.2 (M), 350.2 (100%), 301.2.

[0162] Compound 69c was prepared from 68c as described above for 69a in96% yield. ¹H-NMR (D₂O): δ1.46 (m, 4H, CH₂), 1.78 (m, 4H, CH₂), 3.16 (m,4H, NCH₂), 4.39 (s, 4H, benzylic CH₂), 7.74 (m, 4H, Ph), 7.91 (m, 4H,Ph); ¹³C-NMR (DMSO d): δ25.24, 25.82, 46.73, 49.44, 128.35, 128.56,130.81, 138.38, 165.58. MS-ESI (m/z) 381.2 (M⁺), 191.2 (100%), 150, 116.

[0163] Compound 70: Triamide 18 (4.3 g, 5.8 mmol) was dissolved in 30 mlof DMF and 80% NaH (208 mg, 6.9 mmol) was added. The mixture was stirredunder a N₂ atmosphere for 1 h and 1.12 g (7.5 mmol) ofbromobutyronitrile dissolved in 3 ml of DMF were added all at once. Thereaction mixture was heated for 3 h at 90° C. The solvent wasdistilled-off and the residue was dissolved in chloroform and washedtwice with a saturated solution of amonium chloride; dried (NaSO₄) andevaporated to dryness. Flash chromatography of the residue usinghexane-ethyl acetate (6:4) as eluant gave the yellow oil 70 (3.7 g,77%). ¹H-NMR CDCl₃): δ0.95 (t, 3H), 1.35 (m, 8H), 1.85 (m, 2H), 2.20 (t,2H), 2.30 (s, 9H), 2.55 (s, 18H), 3.10 (m, 10H), 3.25 (t, 2H), 6.95 (s,6H). MS-FAB (m/z) 823 (M⁺+Na), 639, 457.

[0164] Compound 71: Nitrile 70 (3.7 g, 4.6 mmol) was dissolved in 20 mlof chloroform and 150 ml of ethanol were added. The mixture was reducedover 0.35 g of PtO₂ at 50 psi over night. The catalyst was filtered-offand the solvent evaporated to dryness. The oily residue was dried invacuo for 2 h and dissolved in 50 ml of Cl₃CH and 12 ml 2N NaOH. Themixture was cooled in an icewater bath with efficient magnetic stirringand 1.50 g (6.9 mmol) of mesitylene chloride dissolved in 10 ml ofchloroform were added all at once. After 2 h the organic layer wasseparated, washed twice with a saturated solution of amonium chloride,dried (NaSO₄) and evaporated to dryness. Flash chromatography of theresidue using hexane-ethyl acetate (7:3) as eluant provided thetetramide 71 as a colorless oil (3.3 g, 73% over two steps). ¹H-NMRCDCl₃): δ0.95 (t, 3H), 1.40 (m, 12H), 2.30 (s, 12H), 2.60 (s, 24H), 2.80(b, 2H), 3.10 (m, 12H), 4.70 (b, 1H), 6.90 (s, 8H). MS-FAB (m/z) 1010(^(M+)+1+Na), 826, 643.

[0165] Compound 72: The tetramide 71 (6.28 g, 6.3 mmol) was dissolved in40 ml of DMF and 80% NaH (230 mg, 7.6 mmol) was added. The mixture wasstirred under a N₂ atmosphere for 1 h and 1.30 g (8.8 mmol) ofbromobutyronitrile dissolved in 3 ml of DMF were added al] at once. Thereaction mixture was heated for 3h at 90° C., the solvent wasdistilled-off and the residue was extracted into chloroform and washedtwice with a saturated solution of amonium chloride; dried (NaSO₄) andevaporated to dryness. Flash chromatography of the residue withhexane-ethyl acetate (7:3) as eluant provided the nitrile 72 (5.0 g,74%). ¹H-NMR (CDCl₃): δ0.95 (t, 3H), 1.35 (m, 12H), 1.80 (m, 2H), 2.25(t, 2H), 2.35 (s, 12H), 2.70 (s, 24H), 3.10 (m, 14H), 3.25 (t, 2H), 7.0(s, 8H). MS-FAB (m/z) 1077 (M⁺+1+Na), 893, 711, 586.

[0166] Compound 73: Nitrile 72 (6.0 g, 5.6 mmol) was dissolved in 20 mlof chloroform and 150 ml of ethanol were added. The mixture was reducedover 600 mg of PtO₂ at 50 psi overnight. The catalyst was filtered-offand the solvent evaporated to dryness. The oily residue was dried invacuo for 2 h and dissolved in 100 ml of chloroform and 15 ml 2N NaOH.The mixture was cooled in an icewater bath with efficient magneticstirring, and 1.80 g (8.4 mmol) of mesitylene chloride dissolved in 10ml of Cl₃CH was added all at once. After 2 h the organic layer wasseparated, washed twice with a saturated solution of amonium chloride,dried (Na₂SO₄) and evaporated to dryness. Flash chromatography of theresidue using hexane-ethyl acetate (7:3) as eluant gave the pentamide 73as a colorless oil (5.0 g, 71% over two steps). ¹H-NMR (CDCl₃): δ0.95(t, 3H), 1.35 (m, 16H), 2.30 (s, 15H), 2.55 (s, 30H), 2.75 (bs, 2H),3.05 (m, 16H), 4.70 (b, 1H), 6.90 (s, 10H). MS-FAB (m/z) 1261 (M⁺-1+Na),1077, 895.

[0167] Compound 74: Pentamide 73 (3.4 g, 2.7 mmol) was dissolved in 30ml of DMF and 60% NaH (162 mg, 4.05 mmol) was added. The mixture wasstirred under a N₂ atmosphere for 0.5 h and 2.3 g (10.8 mmol) of2-bromoethanol benzylether dissolved in 3 ml of DMF were added all atonce. The reaction mixture was heated for 2 h at 80° C., the solvent wasdistilled-off and the residue was dissolved in chloroform and washedtwice with a saturated solution of amonium chloride, dried (NaSO₄) andevaporated to dryness. Flash chromatography of the residue usinghexane-ethyl acetate (7:3) as eluant provided the product 74 (2.6 g,70%). ¹H-NMR CDCl₃): δ0.95 (t, 3H), 1.30 (m, 16H), 2.30 (s, 15H), 2.50(s, 30H), 2.90-3.20 (m, 18H), 3.25 (t, 2H), 2.35 (t, 2H), 4.35 (s, 2H),6.95 (s, 10H), 7.20-7.35 (m, 5H). ¹³C NMR CDCl₃): δ12.65, 20.84, 22.67,22.71, 24.41, 24.66, 39.97, 44.48, 44.88, 46.59, 68.01, 72.95, 127.46,127.57, 128.25, 131.83, 131.89, 133.28, 139.88, 139.95, 140.04, 142.16,142.23. MS-FAB (m/z) 1394 (M⁺-2+Na) 1030.

[0168] Compound 75: Pentamide 74 (1.2 g, 0.87 mmol) was dissolved in 12ml of methylene chloride followed by the addition of 30% HBr/acetic acid(16 ml) and phenol (3.0 g, 32 mmol). The mixture was stirred at roomtemperature overnight, water (16 ml) was added, followed by extractionwith methylene chloride (3×10 ml). The aqueous layer was evaporated invacuo. The residue was dissolved in 2N NaOH (4 ml) and 50% KOH (4 ml)followed by extraction with chloroform (4×10 ml). After removal of thesolvent the residue was dissolved in ethanol (20 ml) and acidified withconcentrated hydrochloric acid (0.5 ml). The white precipitate (75) wasrecrystallized from aqueous ethanol (440 mg, 90%); mp above 270° C.(decomp). ¹H-NMR (D₂O): δ1.30 (t, 3H), 1.75 (b, 16H), 2.90-3.30 (mn,20H), 2.85 (t, 2H). ¹³C NMR (D₂O): δ13.29, 25.48, 25.59, 45.70, 49.04,49.49, 49.67, 51.88, 59.39. MS-MALDI (m/z) 374 (M⁺+1).

[0169] Compound 76: Pentamide 73 (850 mg, 0.68 mmol) was dissovled inDMF (15 ml) and 80% NaH (30 mg, 1 mmol) was added. The mixture wasstirred under a N₂ atmosphere at room temperature for 0.5 h and 137 mg(0.30 mmol) of 73 dissolved in 5 ml of DMF were slowly added. Thereaction mixture was heated for 2 h at 80° C., the solvent wasdistilled-off and the residue was dissolved in chloroform and washedtwice with a saturated solution of amonium chloride, dried (NaSO₄) andevaporated to dryness. Flash chromatography of the residue usinghexane-ethyl acetate-methanol (6:4:0.1) as eluant afforded the product76 (590 mg, 77%). ¹H-NMR (CDCl₃): δ0.95 (t, 6H), 1.15-1.40 (m, 32H),2.30 (s, 30H), 2.55 (s, 60H), 2.90-3.25 (m, 36H), 3.60 (d, 4H), 5.40 (t,2H), 6.95 (s, 20H). MS-FAB 2553 (MF⁺+Na).

[0170] Compound 77 was obtained following the procedure described forcompound 75. From 650 mg (0.25 mmol) of decamide 76, 225 mg (81 %) ofdecahydrochloride 77 was obtained; mp >270° C. (decomp). ¹H-NMR (D₂O):δ1.30 (t, 6H), 1.75 (b, 32H), 3.10 (b, 36H), 3.75 (b, 4H), 6.05 (b, 2H);¹³C NMR (D₂O): δ13.28, 25.57, 45.66, 49.00,49.13, 49.64, 50.86, 131.15.MS-ESI 711 (M⁺+1).

[0171] Compound 78 was obtained following the procedure described forcompound 76. From 850 mg of 73, 360 mg (47%) of decamide 78 wereobtained. ¹H-NMR (CDCl₃): δ0.95 (t, 6H), 1.15-1.45 (m, 32H), 2.30 (s,30H), 2.55 (s, 60H), 2.90-3.20 (b, 36H), 3.65 (d, 4H), 5.40 (t, 2H),6.90 (s, 20H). MS-FAB (m/z) 2553 (M⁺+Na).

[0172] Compound 79 was obtained following the procedure described forcompound 75. From 330 mg (0.13 mmol) of decamnide 78, 127 mg (90%) ofdecahydrochloride 79 was obtained; mp >270° C. (decomp). ¹H-NMR (D₂O):δ1.30 (t, 6H), 1.80 (b, s, 32H), 3.10 (b, 36H), 3.85 (d, 4H), 6.0 (t,2H). ¹³C NMR (D₂O): δ13.31, 25.59, 45.71, 46.83, 49.05, 49.39, 49.69,129.21. MS-ESI (m/z) 512 (M⁺+2).

[0173] Compound 96. Pentamide 74 (1.4 g, 1.01 mmol) was dissolved in 100mnl of ethanol and 200 mg of 10% Pd/C was added. The mixture washydrogenated for 4 h at 50 psi. The catalyst was filtered off and andsolvent evaporated to dryness. Silica-gel column chromatography usingethyl acetate/hexane 6:4 as running solvent afforded 1.0 g (80%) ofdesired product, as an oil. ¹NMR (CDCl₃) δ: 0.95 (t, 3H), 1.30 (m, 16H),2.30 (s, 15H), 2.55 (s, 30H), 3.10 (m, 18H), 3.25 (t, 2H), 3.60 (t, 2H),6.95, (s, 10H), ¹³C NMR δ: 12.67, 20.89, 22.75, 24.52, 40.02, 44.54,44.97, 46.83, 48.22, 60.29, 131.88, 132.78, 133.28, 139.95, 140.11,142.33

[0174] Compound 97 Alcohol 96 (470 mg, 0.36 nmmole) was dissolved intetrahydrofuran (5 ml), Boc-Gln (97 mg, 0.39 mmole),dicyclohexylcarbodiimide (89 mg, 0.43 mmole), and dimethylaminopyridine(5 mg, 0.039 mmole) were added. The reaction mixture was stirredovernight at room temperature. The cyclohexylurea was filtered off andthe filtrate evaporated to dryness. The residue was dissolved inchloroform, washed twice with 2N HCl, once with water, and twice with asaturated solution of NaHCO₃, then dried and evaporated. The product waspurified by silica-gel column chromatography using methanol/chloroform2% as running solvent. The amino acid-polyamine analog conjugate weighed250 mg (45%). ¹NMR (CDCl₃) δ: 0.95 (t, 3H), 1.30 (m, 18H), 1.45 (s, 9H),1.90-2.20 (m, 211), 2.35 (s, 15H), 2.60 (s, 30H) 2.90-3.25 (m, 18H),3.45 (m, 2H), 4.10-4.35 (m, 3H), 6.95 (s, 10H); ¹³C NMR (CDCl₃) δ:12.57, 20.78, 22.63, 24.63, 28.19, 31.48, 39.92, 44.04, 44.43, 44.82,45;92, 53.06, 61.96, 79.80, 131.99, 133.33, 139.80, 142.12, 156.40,171.70, 174.25.

[0175] Compound 98 Amino acid-polyamine analog conjugate 97 (170 mg,0.11 mmole) was treated with trifluoroacetic acid (1.25 ml) in methylenechloride (5 ml) for 30 minutes. The solvent was evaporated at roomtemperature, the residue was dissolved in chloroform and washed with asaturated solution of NaHCO₃, then dried and evaporated to dryness.After drying in vacuo, the residue weighted 158 mg (100%). The remainingprotecting groups are removed following the procedure described forcompound 21.

Example 2. In vitro activity of polyamine- and polyamine analog-aminoacid conjugates.

[0176] Polyamine- and polyamine analog-amino acid conjugates of theinvention were tested for their ability to inhibit growth of variouscancer cell lines. The procedures used for testing activity are similarto those used in International Patent Application WO 00/66587 and U.S.Pat. No. 5,889,061 and are described below.

[0177] Cell Lines and Media

[0178] Human breast cancer cell line MCF7 was grown in Richter'sImproved Modified Eagle's Medium supplemented with 10% fetal bovineserum (FBS) and 2.2 gIL sodium bicarbonate. Human brain tumor cell lineU251 MG-NCI was grown in Dulbecco's Modified Eagle's Medium supplementedwith 10% FBS. Human lung cancer cell line A549 was grown in Ham's F-12Kmedium (Cellgro, Mediatech, Inc., Va.), supplemented with 10% FBS and 2mM L-glutamine. Human colon cancer cell line HT129 was cultured inMcCoy's 5A medium (Gibco, BRL, Gaithersburg, Md.) supplemented with 10%FBS. Human prostate cancer cell lines PC-3, LNCAP and DuPro were grownin RPMI 1640 Medium (Cellgro, Mediatech, Inc., Va.) supplemented with10% FBS. Another human prostate cancer cell line DU145 was grown inDulbecco's Modified Eagle's Medium (Gibco, BRL, Gaithersburg, Md.)supplemented with 5% FBS. The A549, MCF7, PC3, LNCAP and DuPro celllines were cultured in 100 units/mL penicillin and 100 μg/mLstreptomycin. HT29 and U251MG cell lines were grown in 50 μg/mLgentamycin (Gibco, BRL, Gaithersburg, Md.). DU145 cell line wasmaintained in 1% antibitic-antimycotic solution (Sigma, St. Louis, Mo.)The cell cultures were maintained at 37° C. in 5% CO₂/95% humidifiedair. DuPro cells were obtained from M. Eileen Dolan, University ofChicago. All other cells are available from the American Type CultureCollection, Rockville, Md.

[0179] MTT assay

[0180] A conventional MTT assay was used to evaluate percent cellsurvival. Exponentially growing monolayer cells were plated in 96-wellplates at a density of 500 cells per well and allowed to grow for 24hours. Serial dilutions of the drugs were added to the wells. Six daysafter drug treatment, 25 μl of MTT solution (5 mg/ml) was added to eachwell and incubated for 4 hours at 37° C. Then 100 μl of lysis buffer(20% sodium dodecyl sulfate, 50% DMF, and 0.8% acetic acid, pH 4.7) wasadded to each well and incubated for an additional 22 hours. Amicroplate reader (“EMAX”-brand, Molecular Devices, Sunnyvale, Calif.)set at 570 nm was used to determine the optical density of the cultures.Results are expressed as a ratio of the optical density in drug-treatedwells to the optical density in wells treated with vehicle only.

[0181] The ID₅₀ of two of the compounds of the invention, SL-11143 (apolyamine linked to beta-alanine) and SL-11165 (a polyamine linked toglutamine) against various cancer cell lines are listed in the tablebelow. ID₅₀ ID₅₀ ID₅₀ ID₅₀ Compound Structure DuPro PC-3 DU145 LnCapSL-11143

13.75 >31.25 0.06 6.4 SL-11165

>31.65 4.1 >31.25

[0182] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it is apparent to those skilled in the art that certainminor changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention.

[0183] The following U.S. patent applications are hereby incorporated byreference herein in their entirety: U.S. Provisional Patent ApplicationSer. No. 60/131,809, U.S. Provisional Patent Application Ser. No.60/131,779, and U.S. Provisional Patent Application Ser. No. 60/131,842,all filed on Apr. 30, 1999; and U.S. Ser. No. 09/561,172, U.S. Ser. No.09/560,711, and U.S. Ser. No. 09/562,980, all filed on Apr. 27, 2000.

[0184] All references, patents, patent applications, and non-patentpublications mentioned herein are hereby incorporated by referenceherein in their entirety.

What is claimed is:
 1. A composition comprising a polyamine or polyamineanalog conjugated to at least one amino acid.
 2. The composition ofclaim 1, wherein only one amino acid is conjugated to the polyamine orpolyamine analog, and the amino acid is conjugated at one and only oneof the exterior nitrogens of the polyamine or polyamine analog.
 3. Theconjugate of claim 1, wherein two amino acids are conjugated to thepolyamine or polyamine analog, where each amino acid is conjugated to anexterior nitrogen of the polyamine or polyamine analog.
 4. Thecomposition of claim 1, wherein the polyamine- or polyamine analog-aminoacid conjugates are of the formula:(M)-N(-E)-B-A-B-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH-E or(M)-N(-E)-B-A-B-NH-B-A-B-NH-B-A-B-NH-B-A-B-N(M)-E

wherein each M is independently an amino acid, each A is independentlyselected from the group consisting of a single bond, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆cycloalkenyl; each B is independently selected from the group consistingof: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; and each E isindependently selected from the group consisting of H, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, andC₃-C₆ cycloalkenyl; and any salt or stereoisomer thereof.
 5. Thecomposition of claim 1, wherein the polyamine- or polyamine analog-aminoacid conjugates are of the formula:(M)-N(-E)-B-A-B-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH-E or(M)-N-(-E)-B-A-B-NH-B-A-B-NH-B-A-B-NH-B-A-B-N(M)-E

wherein each M is independently an amino acid, each A is independentlyselected from the group consisting of a single bond, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆cycloalkenyl; each B is independently selected from the group consistingof: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; and each E isindependently selected from the group consisting of H, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, andC₃-C₆ cycloalkenyl; with the proviso that either at least one A moietyis selected from the group consisting of C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆ cycloalkenyl, or at leastone B moiety is selected from the group consisting of C₂-C₆ alkenyl; andany salt or stereoisomer thereof.
 6. The composition of claim 1, whereinthe polyamine- or polyamine analog-amino acid conjugates are of theformula: (M)-N(E)B-A-B-NH-B-A-B-NH-B-A-B-NH(-B-A-B-NH)_(x)-E or(M)-N(-E)-B-A-B-NH-B-A-B-NH- B-A-B-NH(-B-A-B-NH)_((x-1))-(-B-A-B-N(M))E

wherein each M is independently an amino acid, each A is independentlyselected from the group consisting of: a single bond, C₆-C₂ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆cycloalkenyl; each B is independently selected from the group consistingof: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; each E isindependently selected from the group consisting of H, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, andC₃-C₆ cycloalkenyl; and x is an integer from 2 to 16; and any salt orstereoisomer thereof.
 7. The composition of claim 1, wherein thepolyamine- or polyamine analog-amino acid conjugates are of the formula:(M)-N(-E)-B-A-B-NH-B-A-B-NH-B-A-B-NH(-B-A-B-NH)_(x)-E or(M)-HN-B-A-B-NH-B-A-B-NH- B-A-B-NH(-B-A-B-NH)_((x-1))-B-A-B-N(M)-E

wherein each M is independently an amino acid, each A is independentlyselected from the group consisting of: a single bond, C₆-C₂ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆cycloalkenyl; each B is independently selected from the group consistingof: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; each E isindependently selected from the group consisting of H, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, andC₃-C₆ cycloalkenyl; and x is an integer from 2 to 16; with the provisothat either at least one A moiety is selected from the group consistingof C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, andC₃-C₆ cycloalkenyl, or at least one B moiety is selected from the groupconsisting of C₂-C₆ alkenyl; and any salt or stereoisomer thereof. 8.The composition of claim 1, wherein the polyamine- or polyamineanalog-amino acid conjugates are of the formula:(M)E-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH(-B-A-B-NH)_(x)- E(M) or(M)E-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH(-B-A-B-NH)_(x)-E

wherein each M is independently an amino acid, wherein each A isindependently selected from the group consisting of: a single bond,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, andC₃-C₆ cycloalkenyl; each B is independently selected from the groupconsisting of: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; each E isindependently selected from the group consisting of C₁-C₆ alkyl, C₁-C₆alkanol, C₃-C₆ cycloalkanol, and C₃-C₆ hydroxyaryl, and the aminoacid(s) is linked to the polyamine analog via an ester linkage at the Egroup hydroxyl(s), with the proviso that each E bearing an amino acid isselected from C₁-C₆ alkanol, C₃-C₆ cycloalkanol, and C₃-C₆ hydroxyaryl;and x is an integer from 0 to 16; and any salt or stereoisomer thereof.9. The composition of claim 1, wherein the polyamine- or polyamineanalog-amino acid conjugates are of the formula:(M)E-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH(-B-A-B-NH)_(x)- E(M) or(M)E-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH(-B-A-B-NH)_(x)-E

wherein each M is independently an amino acid, wherein each A isindependently selected from the group consisting of: a single bond,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, andC₃-C₆ cycloalkenyl; each B is independently selected from the groupconsisting of: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; each E isindependently selected from the group consisting of C₁-C₆ alkyl, C₁-C₆alkanol, C₃-C₆ cycloalkanol, and C₃-C₆ hydroxyaryl, with the provisothat each E bearing an amino acid is selected from C₁-C₆ alkanol, C₃-C₆cycloalkanol, and C₃-C₆ hydroxyaryl; and the amino acid(s) is linked tothe polyamine analog via an ester linkage to at least one E grouphydroxyl(s); and x is an integer from 0 to 16; and any salt orstereoisomer thereof.
 10. The composition of claim 1, wherein thepolyarnine- or polyamine analog-amino acid conjugates are of theformula: (M)-N(-E)-D-NH-B-A-B-NH-D-NH-E or(M)-N(-E)-D-NH-B-A-B-NH-D-N(M)-E

wherein each M is independently an amino acid; A is selected from thegroup consisting of C₂-C₆ alkynyl; each B is independently selected fromthe group consisting of: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl;each D is independently selected from the group consisting of C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆cycloalkenyl, and C₃-C₆ cycloaryl; and each E is independently selectedfrom the group consisting of H, C₁-C₆ alkyl, C?-C₆ alkenyl, C₂-C₆alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆ cycloalkenyl; andany salt or stereoisomer thereof.
 11. The composition of claim 1,wherein the polyamine- or polyamine analog-amino acid conjugates are ofthe formula: (M)-N(-E)-B-A-B-NH-F-NH-B-A-B-NH-E or(M)-N(-E)-B-A-B-NH-F-NH-B-A-B-N(M)-E

wherein each M is independently an amino acid; F is selected from thegroup consisting of C₁-C₆ alkyl; each A is independently selected fromthe group consisting of: a single bond, C₁-C₆ alkyl; C₂-C₆ alkenyl,C₂-C₆ alk)ynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆cycloalkenyl;each B is independently selected from the group consistingof: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; and each E isindependently selected from the group consisting of H, C₁-C₆ alkyl,('2-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, andC₃-C₆ cycloalkenyl; and any salt or stereoisomer thereof.
 12. Thecomposition of claim 1, wherein the polyamine- or polyamine analog-aminoacid conjugates are of the formula: (M)-N(-E)-B-A-B-NH-F-NH-B-A-B-NH-Eor (M)-N(-E)-B-A-B-NH-F-NH-B-A-B-N(M)-E

wherein each M is indepedently an amino acid; wherein F is selected fromthe group consisting of C₁-C₆ alkyl; each A is independently selectedfrom the group consisting of: a single bond, C₁-C₆ alkyl; C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆cycloalkenyl; each B is independently selected from the group consistingof: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; and each E isindependently selected from the group consisting of H, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, andC₃-C₆ cycloalkenyl; with the proviso that either at least one A moietyis selected from the group consisting of C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆ cycloalkenyl, or at leastone B moiety is selected from the group consisting of C₂-C₆ alkenyl; andany salt or stereoisomer thereof.
 13. A composition according to any oneof claims 1-12, wherein the amino acid is chosen from the set of allamino acids with either amide-containing or basic side chains, and allstereoisomers and salts thereof.
 14. A composition according to any oneof claims 1-12, wherein the amino acid is chosen from glutamine,asparagine, lysine, ornithine, arginine, histidine, or citrulline, andall stereoisomers and salts thereof.
 15. A composition according to anyone of claims 1-12, wherein the amino acid is chosen from D-glutamine orL-glutamine, and all salts thereof.
 16. A composition according to anyone of claims 1-12, wherein the amino acid is chosen from L-glutamine,and all salts thereof.
 17. A composition according to any one of claims1-12, wherein the amino acid is an alpha-amino acid, and is linked tothe polyamine analog via an amide linkage between the alpha-carboxylgroup of the amino acid and a nitrogen of the polyamine analog.
 18. Acomposition comprising a polyamine analog-amino acid conjugate of theformula: CH₃CH₂-N(R₁₀₀)-J-N(R₁₀₀)-CH₂CH₃ where each R₁₀₀ isindependently chosen from H, C₁-C₈ alkyl, and an amino acid, with theproviso that at least one R₁₀₀ be an amino acid; and where J is selectedfrom {C₁-C₈ alkyl-[N(R₁₀₀)-(C₁-C₈ alkyl)]_(k)}, where each R₁₀₁ isindependently selected from H and C₁-C₈ alkyl, and where k is an integerbetween 0 and
 15. 19. The composition of claim 18, wherein one and onlyone R₁₀₀ is an amino acid.
 20. The composition of claim 18, wherein oneand only one R₁₀₀ is an amino acid and the other R₁₀₀ is H.
 21. Thecomposition of claim 18, where the amino acid is chosen from amino acidswith either amide-containing or basic side chains, and all stereoisomersand salts thereof.
 22. The composition of claim 18, where the amino acidis chosen from glutamine, asparagine, lysine, ornithine, arginine,histidine, or citrulline, and all stereoisomers and salts thereof. 23.The composition of claim 18, where the amino acid is D-glutamine orL-glutamine, and all salts thereof.
 24. The composition of claim 18,where the amino acid is L-glutamine, and all salts thereof.
 25. Acomposition comprising a polyamine analog-amino acid conjugate of theformula:CH₃CH₂-N(R₁₀₀)-(CH₂)₄-N(R₁₀₁)-(CH₂)₄-N(R₁₀₁)-(CH₂)₄-N(R₁₀₁)-(CH₂)₄-N(R₁₀₀)-CH₂CH₃where each R₁₀₀ is independently chosen from H, C₁-C₈ alkyl, and anamino acid, with the proviso that at least one R₁₀₀ be an amino acid;and each R₁₀₁ is independently chosen from H and C₁-C₈ alkyl.
 26. Acomposition comprising any of the compositions of claims 1-25, furthercomprising a pharmaceutically acceptable excipient or carrier.
 27. Amethod of treating cancer in an individual, comprising administering tothe individual an effective amount of a composition of claims 1-26.